Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps in Dengue Are Mainly Generated NOX-Independently

doi: 10.3389/fimmu.2021.629167

Figure Lengend Snippet: Platelets stimulate NOX-independent NET formation in a DENV NS1-dependent manner. Polymorphonuclear neutrophils (PMNs) of healthy volunteers were co-incubated with autologous platelets in the presence or absence of 10 µg/mL DENV2 NS1 or 156μM platelet agonist TRAP-6. Experiments were performed in triplicate with 2 donors for each condition. Depicted are the relative values to medium (unstimulated samples) for (A) Total NETs and (B) NOX-independent NETs. (C) Heat-inactivated DENV2 does not induce NET formation. PMNs isolated from three healthy donors were incubated with 0.5x10 7 TCID/mL heat-inactivated DENV2 or medium control (mock) in the presence or absence of autologus washed platelets (Plt). Depicted is the total NET formation (ng/mL). Data are presented as mean with standard error mean (SEM) and analyzed using repeated measure a one-way ANOVA and Bonferroni’s multiple comparisons test; * p < 0.05, **p < 0.01 . TCID, Tissue culture infectious dose.

Article Snippet: After 1h, cells were washed with pre-warmed PBS and co-cultured with or without washed platelets (ratio 1 PMN vs . 50 platelets) in the presence of the following stimuli: 10µg/mL Dengue virus-2 non-structural protein-1 (DENV2 NS1, strain Thailand/16681/84; Native Antigen, Oxford, UK) or 156 µM Thrombin Receptor Activator Peptide 6 (TRAP-6) (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 3 hours at 37°C.

Techniques: Incubation, Isolation

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 2. Characterization of the NS1– population during MNoV infection. (A) Depiction of the NS1 positivity and negativity in CR6-infected BV2 cells. (B) Intracellular flow cytometry plots of CR6-infected BV2 cells costained with NS6/7 and NS1 (measured with clone CM79) at 16 hpi. (C) Intracellular flow cytometry plots of CR6-infected BV2 cells costained with NS6/7 and NS1 (measured with clone 2A9) at 8 or 18 hpi. Q-VD-OPh (20 μM) was added along with the virus when indicated. (D and E) Intracel- lular flow cytometry plots of WT or Casp3 KO BV2 cells infected with CR6 (D) or BV2 cells infected with WT CR6 or CR6D121/131G mutant virus (E) and costained with NS6/7 and NS1 at 18 hpi. (F) Comparison of caspase-3 activation in NS1– and NS1+ cells, measured with an antibody targeting cleaved caspase-3. (G) Surface staining with an- nexin V compared in NS1– and NS1+ cells. (H) BV2 cells were infected with CR6, harvested at 18 hpi, and costained with NS1, NS6/7, and other viral proteins. Intracellular levels of indicated viral proteins measured by flow cytometry in NS1– and NS1+ cells are shown.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Infection, Flow Cytometry, Virus, Mutagenesis, Comparison, Activation Assay, Staining

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 3. NINJ1-mediated membrane rupture is essential for NS1 secretion. (A) Volcano plot showing sgRNAs depleted or enriched in NS1– cells compared to NS1+ cells. (B to E) WT or Ninj1 clonal KO BV2 cells were infected with MNoV CR6 at 5 multiplicity of infection (MOI). (B) NS1 protein levels in the supernatant were measured at 14 hpi by enzyme-linked immunosorbent assay (ELISA) (n = 3). (C) LDH release was measured at 16 hpi (n = 3). (D) Sytox uptake was measured at 12 ~ 20 hpi (n = 3). (E) Cells (WCL) and supernatants (Sup) were analyzed by immunoblotting at 10 ~ 16 hpi. (F and G) WT or Ninj1 KO iBMDMs (F) or IEC-M2C-CD300lf cells transduced with indicated sgRNAs (G) were infected with CR6, and NS1 secretion was measured at 14 hpi (n = 3). (H to K) WT or Ninj1 KO BV2 cells were transduced with NINJ1 WT or oligomerization- defective mutant (K45Q) and infected with CR6. (H) Cells and supernatants were analyzed by immunoblotting at 14 hpi. (I) NS1 secretion was measured by ELISA at 14 hpi (n = 6). (J) Sytox uptake was measured at 14 ~ 20 hpi (n = 3). (K) LDH release was measured at 16 hpi (n = 3). (L) WT or Ninj1 KO BV2 cells were infected with CR6 and treated with glycine (5 mM). NS1 secretion was measured at 14 hpi (n = 3). (M) BV2 cells were infected at 1 MOI with WT CR6 or mutant virus (CR6∆N) and analyzed by ELISA and immunoblotting at 10 hpi (n = 6 from two independent experiments). Data represent means ± SEM. Data were normalized to Tween-20–treated control [(C), (D), (J), and (K)]. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test [(B), (C), (F), (G), and (I)] and Tukey’s multiple comparisons test [(K) and (L)] or two-tailed unpaired Mann-Whitney test (M). ns, not significant; **P < 0.01; ****P < 0.0001. Data are representative of three independent experiments.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Membrane, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Transduction, Mutagenesis, Virus, Control, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 4. Amino acid residues within NS1 required for the specific secretion of NS1. (A) Mutant studies revealed residues required for NS1 secretion. (B) BV2 cells were transfected with a mock vector or 2xStrep-NS1/2 and caspase-3 activation was measured by flow cytometry. (C) WT or Ninj1 KO BV2 cells were transfected with 2xStrep- NS1/2. Cells and supernatants were harvested at 24 hours posttransfection and analyzed by immunoblotting. (D) BV2 cells were transfected with 2xStrep-tagged NS1/2 constructs with N-terminal truncation mutations, and cells and supernatants were analyzed by immunoblotting at 24 hours posttransfection. Red arrowheads indicate different truncation mutants. (E) BV2 cells were transfected with D121/131G cleavage mutant and triple-alanine scanning mutants and analyzed as in (D). Numbers indi- cate amino acid residues substituted by alanine. Mutants showing defect in secretion are highlighted in red. (F) BV2 cells were transfected with NS1/2 constructs with indicated point mutations and analyzed as in (D). (G) 3D structure of CR6 NS1 (58 to 114 amino acids; PDB: 2MCK) highlighting the key residues identified from mutagen- esis study. (H and I) BV2 cells with doxycycline-inducible expression of 2xStrep-tagged WT or mutant NS1/2 were infected with MNoV CR6 and incubated with doxycycline (2 μg/ml) for 12 hours. (H) Cells (WCL) and supernatants (Sup) were analyzed by immunoblotting. (I) LDH release was measured and normalized to Tween-20–treated control (n = 3). Data represent means ± SEM and were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Activation Assay, Flow Cytometry, Western Blot, Construct, Expressing, Infection, Incubation, Control

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 5. NS1 directly binds to NINJ1. (A) WT or Ninj1 KO BV2 cells were infected with CR6 at 5 MOI and harvested at 10 hpi. Cell lysates were immunoprecipitated with anti- NS1 and analyzed by immunoblotting. (B and C) Transmission electron microscopy image of uninfected (B) or CR6-infected (C) BV2 cells with immunogold labeling for FLAG-NINJ1 (12-nm gold, black arrowheads) and NS1 (18-nm gold, red arrowheads). RC, replication complex. Scale bar, 500 nm. (D) Representative immunofluorescence confocal imaging of FLAG-NINJ1 (red), NS1 (green), NS6/7 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI, blue) in Ninj1 KO BV2 cells reconstituted with FLAG-NINJ1 WT or K45Q at 14 hpi. White arrowhead highlights the colocalization of NINJ1 and NS1 proteins. Scale bars, 5 μm. All images are representative of three or more independent ex- periments. (E) Quantification of colocalization between NS1 and FLAG-NINJ1 WT or K45Q. Bars represent means ± SEM, n = 10 to 11 cells. (F) Dose-dependent binding of Fc-tagged ectodomain of human NINJ1 (hNINJ1) or mouse NINJ1 (mNINJ1) protein to recombinant NS1 protein was determined by ELISA. A monoclonal anti-NS1 (clone CM79) and isotype control were included as a positive and negative control, respectively. Data represent means ± SEM of duplicates, and the dissociation constants (Kd) are shown. (G to I) Direct binding between recombinant NS1 and mNINJ1 protein was measured by MST. ΔFnorm indicates the change in thermophoresis, and Kd is shown. n.d., not determined. (G) Dose-response curve showing bindings of WT NS1 protein to fluorescence-labeled WT or mutated mNINJ1 proteins. (H) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled mNINJ1. Maltose-binding protein (MBP) was included as a negative control. (I) Dose-response curve show- ing bindings of WT or mutated NS1 proteins to fluorescence-labeled monoclonal anti-NS1 or isotype control. Data are shown in means ± SEM (n = 3).

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Infection, Immunoprecipitation, Western Blot, Transmission Assay, Electron Microscopy, Labeling, Immunofluorescence, Imaging, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Fluorescence

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 6. HSP90 inhibitors prevent NS1 secretion by blocking caspase-3 activation. (A) BV2 cells were infected with CR6 at 5 MOI in the presence of 2321 FDA-approved drugs (dots represent individual drugs tested). NS1 levels in the supernatant were measured by ELISA at 16 hpi and normalized to vehicle control. Representative results from two independent experiments are shown. (B to D) Cells were infected with CR6 in the presence of serial dilutions of drugs. NS1 secretion was measured by ELISA at 16 hpi and normalized to no-drug control. IC50 values (nM) determined by four biological replicates are shown. BV2 cells were treated with 17-AAG (left), AT13385 (middle), or ganetespib (right) (B). IEC-M2C-CD300lf (C) or IEC-Mx2LUC-CD300lf (D) cells were treated with ganetespib. (E) Caspase-3 activation in CR6-infected BV2 cells with gane- tespib (10 μM) was measured with an antibody targeting cleaved caspase-3. For (B) to (E), data are representative of two to three independent experiments. (F to H) WT or Ifnlr1−/− B6 mice were infected with 106 PFU CR6 perorally. Ganetespib was given intravenously (50 mg/kg, iv) as experimental treatment scheme (F). MNoV genomes in stool (G), ileum, colon, and MLN (H) at indicated time points were quantified by qRT-PCR (n = 7 to 10 mice per group, combined from two independent experiments). Data represent means ± SEM and analyzed by two-tailed unpaired Mann-Whitney test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Blocking Assay, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Suspension multiplex immunoassay for IgM reactivity with tick-borne encephalitis whole virus vs non-structural protein 1 antigens in infected (n = 50) vs vaccinated individuals (n = 50) at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Suspension, Multiplex Assay, Virus, Infection

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Tick-borne encephalitis virus reactivity of sera from acutely infected patients (n = 50) vs vaccines (n = 50), at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Virus, Infection, Vaccines

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Suspension multiplex immunoassay for IgG reactivity with tick-borne encephalitis whole virus vs non-structural protein 1 antigens in infected (n = 50) vs vaccinated individuals (n = 50) at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Suspension, Multiplex Assay, Virus, Infection

Journal: Stem Cell Reports

Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

doi: 10.1016/j.stemcr.2016.07.012

Figure Lengend Snippet: HLCs Supported Persistent and Productive Infection of DENV (A) DENV2 infection of HLCs in a differentiation stage-dependent manner. The hESCs (day 0) and differentiated cells at various differentiation stages from day 1 to day 13 were inoculated with DENV2 (strain 16681) at an MOI of 2 for 48 hr, and cell lysates were collected for western blotting. (B) Immunostaining analysis of DENV2-infected HLCs. hESCs (day 0), definitive endoderm (day 3), hepatic progenitors (day 6), and HLCs (day 10) were infected by DENV2 as described above. Cell were fixed and stained with anti-NS3 antibody. Scale bar, 50 μm. (C) DENV2 infection was blocked in HLCs by inhibitors. HLCs at day 15 after differentiation were pretreated with IFN-α2a (1,000 U/mL), MPA (1 μM), CQ (5 μg/mL), or DMSO (0.1%, v/v) for 8 hr, then subjected to DENV2 infection as described above. (D) qRT-PCR analysis of DENV RNA levels. Cells were treated as described above. The relative DENV RNA level in each sample was normalized to DMSO-treated samples. (E) Persistent replication of DENV2 in HLCs. Day-15 HLCs were exposed to DENV2 for 6 hr before the cells were stringently washed. NS1 protein in the supernatant collected every 24 hr after infection was measured by ELISA assay. (F) DENV2 particles released from HLCs efficiently infected C6/36 cells. HLCs at day 15 were infected with DENV2 at an MOI of 2 for 6 hr. The inoculum was then removed and the cells washed stringently with PBS five times before the fresh medium was added. The supernatants were collected every 24 hr from day 16 to day 20. After each change of the spent medium, cells were washed stringently as described above. Each medium supernatant was added to naive C6/36 cells and incubated for 24 hr. Cells were then lysed and subjected to western blotting analysis. DENV2 derived from C6/36 cells was used as a positive control. (G) Virus titration. Virus titers were calculated in the above supernatants collected at 24, 48, and 72 hr post infection. (H) Upregulation of DENV entry factors during the differentiation process. Total RNA from differentiated cells at the indicated days were extracted and subjected to real-time qRT-PCR to measure the cellular expressions of AXL , HAVCR1 , PROS , and CLDN1 . GAPDH was used as the reference and its expression was set as 1. All the genes were normalized to GAPDH to obtain their relative gene-expression ratio. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S2 .

Article Snippet: The supernatants were collected each day from day 16 to day 21 for measuring nonstructural protein 1 (NS1) in the supernatant, using a DENV NS1 Antigen ELISA Kit (Abnova).

Techniques: Infection, Western Blot, Immunostaining, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay, Positive Control, Virus, Titration, Expressing, Gene Expression

Journal: Stem Cell Reports

Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

doi: 10.1016/j.stemcr.2016.07.012

Figure Lengend Snippet: Interferon Response Induced by DENV Infection in HLCs (A) Upregulation of IFITM1 and USP18 upon DENV2 infection. The hESCs (day 0) and differentiated cells from day 1 to day 11 were inoculated with or without DENV2, and cell lysates were collected 48 hr post infection for western blotting. IFN-α2a-treated cells at day 12 were used as a positive control. (B) Inhibition of IFN signaling pathway enhanced DENV2 infection. HLCs at day 15 were pretreated with DMSO (0.1%), JAK inhibitor (1 μM), or TBK1 inhibitor (2 μM) for 3 hr, followed by incubation with or without IFN-α2a (1,000 U/mL) for 8 hr. Cells were then infected with DENV2 at an MOI of 0.3 for 48 hr. Cell lysates were collected for western blotting. (C) Inhibition of IFN signaling resulted in the spread of DENV2. Cells were treated as described above, and infected by DENV2 at an MOI of 0.01 for 72 hr. Cells were fixed and stained for immunostaining. Scale bar, 50 μm. (D) Statistical analysis of the percentage of DENV infection. HLCs were pretreated with DMSO, JAKi, and IFN as described above, then infected by DENV2 at an MOI of 0.5 for 48 hr. The percentage of DENV infection in each sample was analyzed by immunostaining assay. (E–G) Comparison of virus infection between HLCs and Huh 7.5 cells. Both HLCs and Huh 7.5 cells were infected by DENV2 at an MOI of 1 for 48 hr. Cell samples and the supernatants were collected every 24 hr for immunostaining analysis (E), infection rate calculation (F), and virus titration (G). Cells were washed stringently before medium change. Scale bar, 50 μm. (H) No induction of IFITM1 in Huh 7.5 cells upon DENV2 infection. Cells were treated as described above. Cell samples were collected at 24 hr post infection for western blotting. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S3 .

Article Snippet: The supernatants were collected each day from day 16 to day 21 for measuring nonstructural protein 1 (NS1) in the supernatant, using a DENV NS1 Antigen ELISA Kit (Abnova).

Techniques: Infection, Western Blot, Positive Control, Inhibition, Incubation, Staining, Immunostaining, Comparison, Virus, Titration

Journal: Stem Cell Reports

Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

doi: 10.1016/j.stemcr.2016.07.012

Figure Lengend Snippet: DENV Infection Activated NF-κB Signaling Pathway (A) Phosphorylated RELA/p65 entered the nuclei after DENV2 infection. HLCs were infected by DENV2 at an MOI of 2 for 48 hr and then subjected to staining. Scale bar, 50 μm. (B) Statistical analysis of the ratio of phospho-NF-κB RELA/p65 positive cells in the whole population. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗∗∗ p < 0.001. (C) DENV2 infection increased ROS production. HLCs were infected by DENV2 at an MOI of 2 for 48 hr during which time the cells were treated with or without PDTC as an antioxidant (10 μM). General oxidative stress indicator was used to detect ROS production. Scale bar, 50 μm. (D) Transcriptional activation of cytokine and chemokine genes by DENV infection. Bar plot of log 2 (DENV2-treated/untreated) ratios of RNA-seq expression values for cytokines and chemokines targeted by NF-κB signaling ( http://www.bu.edu/nf-kb/gene-resources/target-genes/ ). Bars are colored based on the significance of differential expression based on DESeq2. Note that some genes displayed apparently large differences in expression levels, but lack enough sequencing reads to support a significant change in expression, and these bars are colored black.

Article Snippet: The supernatants were collected each day from day 16 to day 21 for measuring nonstructural protein 1 (NS1) in the supernatant, using a DENV NS1 Antigen ELISA Kit (Abnova).

Techniques: Infection, Staining, Activation Assay, RNA Sequencing, Expressing, Quantitative Proteomics, Sequencing

Journal: Stem Cell Reports

Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

doi: 10.1016/j.stemcr.2016.07.012

Figure Lengend Snippet: Innate Immunity Protected HLCs from Massive Apoptosis Induced by DENV (A) DENV2 infection led to apoptosis of HLCs. At 48 hr post DENV infection at an MOI of 2, HLCs were fixed for TUNEL assay. For the positive control, 0.5 μM doxorubicin, as an apoptosis inducer, was incubated with HLCs for 24 hr. Scale bar, 50 μm. (B) DENV2 infection resulted in PARP and CASP3 cleavage. HLCs at day 15 were pretreated with JAKi (1 μM) or Z-Vad (50 μM) for 3 hr, followed by DENV2 infection as described above. (C) Most of the DENV2-infected cells survived the infection. Day-15 HLCs were incubated with or without JAKi (1 μM) for 3 hr, then subjected to DENV2 infection at an MOI of 5 for 144 hr. Cell samples were collected at different time points after infection for immunostaining. Scale bar, 50 μm. (D) The time course of apoptosis induced by DENV2. HLCs treated with or without IFN-α2a (1,000 U/mL) or JAKi (1 μM) were incubated with or without DENV2 at an MOI of 5 for 144 hr as described above. Cell samples were collected every 24 hr and subjected to MTT assay. Relative cell viability was calculated by comparing each sample with the mock. (E) PARP and CASP3 cleavage during DENV2 infection. HLCs were infected by DENV2 at an MOI of 5 for 144 hr. Cell samples at different time points were collected for western blotting. (F) Comparison of cell death between HLCs and Huh 7.5 cells after DENV2 infection. Both HLCs and Huh 7.5 cells were infected by DENV2 at an MOI of 5 for 96 hr. Cell samples were collected every 24 hr for MTT assay. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 .

Article Snippet: The supernatants were collected each day from day 16 to day 21 for measuring nonstructural protein 1 (NS1) in the supernatant, using a DENV NS1 Antigen ELISA Kit (Abnova).

Techniques: Infection, TUNEL Assay, Positive Control, Incubation, Immunostaining, MTT Assay, Western Blot, Comparison

Journal: Stem Cell Reports

Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

doi: 10.1016/j.stemcr.2016.07.012

Figure Lengend Snippet: DENV Infection Inhibited the Expression of ALB and F5 (A) Bar plot of log 2 (DENV2-treated/untreated) ratios of RNA-seq expression values for the 291 RefSeq genes preferentially expressed in liver ( Liu et al., 2008 ). RNA-seq data were analyzed in the same way as described in Figure 4 . (B) Immunostaining of DENV2-infected HLCs. HLCs at day 15 were infected by DENV2 at an MOI of 2 for 72 hr. Cells were fixed for immunofluorescence analysis. Scale bar, 50 μm. (C) ALB and F5 expression were downregulated in both the HLCs and PHHs after DENV2 infection. PHHs and HLCs were infected by DENV2 at an MOI of 5 for 72 hr and then subjected to western blotting. (D) Other three serotypes of DENV reduced F5 and ALB expression in the HLCs and PHHs. HLCs and PHHs were infected by DENV type 1-Hawaii, DENV type 3-MK-594-87, DENV type 4-H241, and DENV type 4-D85-019 as described above. Cell lysates were collected for western blotting. See also Figure S5 and .

Article Snippet: The supernatants were collected each day from day 16 to day 21 for measuring nonstructural protein 1 (NS1) in the supernatant, using a DENV NS1 Antigen ELISA Kit (Abnova).

Techniques: Infection, Expressing, RNA Sequencing, Immunostaining, Immunofluorescence, Western Blot