EMBO/SourceData huggingface dataset:
Figure 5 C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively). D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).  H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.
Figure 7 B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).
Figure 8  H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.
Figure 9 A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8). B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).
Figure 2LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 hr) (A, B) IL-1β secretion levels were determined in culture media (A, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) (B, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (C, D) IL-1β secretion levels were determined in culture media C, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) D, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or infected with F. tularensis (18 hr) (E, F) IL-1β secretion levels were determined in culture media E, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) F, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with Flatox (3 hr) (G, H). IL-1β secretion levels were determined in culture media G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).
Figure 3LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (B) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with ATP (C) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.(E) LPS-primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.(F) B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.
Figure 5LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30) (A) * with Western blots indicates non-specific bands.LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) (B, PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDHLPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDHLPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDHLPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDHLPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with TcDA (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH H).
Figure 6Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM.(B) Representative confocal micrographs of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM).Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (C, Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (E) Cell death was measured by LDH assay 3 h post-infection. Data information: In E, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (F) IL-1β secretion levels were determined by Luminex assay 1 hr post-infection. Data information: In F, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: In H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).
Figure 1(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)B) Representative images of HRMECs (B; n=4) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. E) Quantification of tube surface area of micrographs shown in B). (E: **P=0.0013 (- vs COCO 30ng/ml); ***P=0.00026 (- vs COCO 60ng/ml)).(C) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO. Scale bar, 500 μm. (F) Quantification of sprouting surface area of micrographs shown in (C; n=6).(G) COCO decreases scratch wound migration after 18h. Scale bar, 150 μm. (H) Quantification of wound closure.(I) COCO decreases EdU incorporation in cultured HUVECs. (J) Quantification of EdU incorporation.(K) COCO does not induced apoptosis in HUVECs. (L) Quantification of apoptotic (Annexin V-positive) and viable (Annexin V-negative) cells (n= 4).
Figure 2(B) Retinal flat mounts of P5 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0059 (PBS vs COCO); *P=0.0169 (PBS vs Flt1Fc). Vessel junctions: **P=0.0063 (PBS vs COCO); *P=0.0159 (PBS vs Flt1Fc); (n=4 mice/group).(D) COCO does not increase empty collagen IV sleeves. Scale bar, 50 μm. (E) Quantification of % of IsoB4+ vessels to Coll IV+ vessels; (n=9).(F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8).(H) COCO injections (P1) do not result in apoptosis in P5 retinas. Scale bar, 40 μm. (I) Quantification of apoptotic cells in control and COCO-injected mice (n=3).
Figure 3(B) Retinal flat mounts of P28 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0019 (PBS vs COCO); *P=0.0128 (PBS vs Flt1Fc). Vessel junctions: **P=0.0073 (PBS vs COCO); *P=0.0302 (PBS vs Flt1Fc); (n=4).(D) COCO injections (P1, P7 and P14) do not result in ONL thinning in P28 retinas. Scale bar, 40 μm. (E) Quantification of apoptotic cells (cleaved caspase-3; CC3) in control and COCO-injected mice (p-values are versus PBS treatment). (n=4).
Figure 4(B) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints; (n=4).(E) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4. Scale bar, 90 μm. (F) Quantification of vascular length and number of branchpoints; (n=4).
Figure 5(B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts.(C) Quantification of neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6).(F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group).
Figure 6(A) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min. Immunoblots are representative of 4 independent experiments. (B) Densitometric analysis of western blot experiments. * P=0.0457; (n=4).(C) COCO treatment does not alter VEGF receptor expression in HUVECs. Immunoblots are representative of 3 independent experiments. (D) Densitometric analysis of western blot experiments; (n=3).
Figure 7(A) Western blot of whole adult human retina extracts incubated with an anti-human COCO antibody and revealing a unique band at ~36 kDa (arrow). Recombinant human COCO was used as positive control.(B) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti-mouse COCO antibody and revealing a unique band at ~36-38 kDa (arrow). Western blots are representative of 3 independent experiments.(C) Immunofluoresence analysis of adult human retina sections with an anti-mouse COCO antibody. Specific immuno-reactivity was observed in multiple areas (arrows) when compared with sections only exposed to the secondary antibody.(D) RNAscope in situ hybridization (Dand5, top; Negative probe (dapB); down) and hematoxylin staining of P5 mouse retinas.(E) Dual RNAscope in situ hybridization and visual-arrestin immunohistochemistry of P5 mouse retinas. Images are representative of 4 animals.(F) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation. Exogenously added COCO was detected using an anti-human COCO antibody, showing co-localization with human mitochondria (MTCO1 antibody).
Figure 8(B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group).
Figure 9(A) Seahorse analysis of mitochondrial respiration (OCR) and glycolysis (ECAR) in HUVECs stimulated with COCO for 1, 2 and 24 hours. **P=0.0032 for ECAR, 1hour; **P=0.0079 for ECAR, 2hours; *P=0.0255 for OCR, 2hours; (n=5).(B) Media glucose levels (BioNova analysis) in HUVECs cultured with COCO for 1 or 6 hours, expressed as % of initial levels *P=0.0459; (n=4).(C) Evaluation of ATP content in HUVECs stimulated with COCO for 1, 6 or 24 hours. *P=0.0120 (- vs 1hour); *P=0.0109 (- vs 6hours); *P=0.0219 (- vs 2-DG); (n=4).(D) Determination of NAD+/NADH ratio in NADH for 6 or 24 hours with COCO. **P=0.0028 (6hours); *P=0.0207 (24hours); (n=3).E) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1, 3, 6, 12 or 24 hours. *P=0.0244; (n=4).(F) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and/or N-acetylcystein. *P=0.0343 (- vs 48hours-NAC); *P=0.0114 (- vs H2O2-NAC); *P=0.0252 (48hours-NAC vs 48hours+NAC); **P=0.0067 (H2O2-NAC vs H2O2+NAC); (n=4).(G) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO. *P=0.0372 (- vs 24hours); *P=0.0137 (- vs 3-NP); (n=3).(H) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and/or N-acetylcystein. Scale bar, 500 μm. (I) Quantification of sprouting surface area of micrographs shown in (H). *P=0.0126 (- vs COCO-NAC); *P=0.0200 (COCO-NAC vs COCO+NAC); (n=6).
Figure 1A. A549 cells were transfected with 2 μg of poly(I:C) for 6 hrs and then infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment) or infected with ZIKV first for 6 hrs and then transfected with poly(I:C) for 12 hrs (Post-treatment). Levels of ZIKVgenomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. ***P<0.001 (Student's t-test), N=3B. A549 cells infected with ZIKV (MOI=3) for 6 hrs were transfected with 2 μg poly(I:C) for 12 hrs after which levels of ifn-C. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2D. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later they were transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test), N=4E. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2F. HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 0.4 μg of poly(I:C). Twenty-four hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3
Figure 2A. A549 cells were treated with IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 6 hrs after which they were infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment). Alternatively, cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs (Post-treatment). Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3B. A549 cells were infected with ZIKV (MOI=3) for 6 hrs prior to addition of IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 12 hrs. Total RNA was isolated and levels of ifit1 RNAs were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3C. A549 cells were infected with ZIKV (MOI=5) for 6 hrs and then transfected with ISRE- or GAS- promoter-driven firefly luciferase together with a constitutively active renilla luciferase construct. Sixteen hours later, cells were treated with IFN-α (100 U/ml) or IFN-α (10 U/ml) for 2 hrs. Cells lysates were harvested and relative luciferase activity from ISRE- and GAS-promoters were determined. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3D. HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK). After 24 hrs they were treated with IFN-α (100 U/mL) for 10 hrs. Cell lysates were then harvested and subjected to luciferase assays. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3
Figure 3A. A549 cells were infected with ZIKV (MOI=5) for 24-48 hrs after which cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.B. A549 cells were infected with ZIKV (MOI=5) for 24 hrs and then treated with DMSO, MG132 (20 µM) or epoxomicin (Epox) (400 nM) for 12 hrs. Cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.C. A549 cells (MOI=2) or Human primary fetal astrocytes (HFA) (MOI=5) were infected with ZIKV for 24 hrs and 48 hrs respectively, after which IFN-α (100 U/mL) was added for 2 hrs. Cells were then fixed and processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. Dashed line white circles indicated ZIKV-infected cells. Representative panels are shown. The experiment was repeated two times.D. Mouse embryonic fibroblasts (MEF) were infected with ZIKV (MOI=5) for 48 hrs after which IFN-α (200 U/mL) was added for 2 hrs. Samples were processed for immunofluorescence assay as described in panel C. Representative panels are shown. The experiment was repeated two times.E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The ZIKV replication was measured by qRT-PCR.E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times.