tak 779  (MedChemExpress)

Journal: Genes to Cells

Article Title: Treatment With Ruxolitinib and TAK ‐779 Enhances GII .17 Human Norovirus Replication and Enables Serial Passaging in Human Intestinal Enteroids

doi: 10.1111/gtc.70139

Figure Lengend Snippet: Treatment with ruxolitinib and TAK‐779 enhances GII.17 replication in HIEs. J2 monolayers were pretreated with ruxolitinib (5 μM) and/or TAK‐779 (30 μM) for 3 h and then inoculated with a stool filtrate containing GII.17 (1.6 × 10 5 GEs/well) in a differentiation medium supplemented with the corresponding compounds and 500 μM GCDCA for 1 h at 37°C. After washing, the cells were cultured with the specified compounds and GCDCA. The cells and culture supernatants were collected at 24, 48, and 96 hpi, and viral RNA was quantified by RT‐qPCR. Differentiation medium containing DMSO and 500 μM GCDCA served as the vehicle control. Data are presented as the mean ± SD ( n ≥ 5). Statistical significance was assessed using the Kruskal–Wallis test followed by Steel's multiple‐comparison test. Asterisks indicate statistically significant differences compared with DMSO‐treated controls at each time point (* p < 0.05). ns, not significant; hpi, h postinfection.

Article Snippet: Briefly, J2 monolayers were pretreated with either ruxolitinib (5 μM; T1829, TargetMol, Boston, MA, USA), TAK‐779 (30 μM; T7499, TargetMol), both, or DMSO (control) at 37°C under 5% CO 2 for 3 h, and then inoculated with HuNoV filtrates—1.6 × 10 5 GEs/well for GII.17, 1.1 × 10 7 GEs/well for GII.4, and 4.3 × 10 5 GEs/well for GII.3—in a differentiation medium supplemented with 500 μM sodium glycochenodeoxycholate (GCDCA; Sigma‐Aldrich) with DMSO or the indicated compounds at 37°C for 1 h. The monolayers were then washed twice with complete medium without growth factors (CMGF[−]).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Comparison

Journal: Genes to Cells

Article Title: Treatment With Ruxolitinib and TAK ‐779 Enhances GII .17 Human Norovirus Replication and Enables Serial Passaging in Human Intestinal Enteroids

doi: 10.1111/gtc.70139

Figure Lengend Snippet: Effects of ruxolitinib and TAK‐779 on GII.3 and GII.4 HuNoV replication in HIEs. J2 monolayers pretreated with ruxolitinib (5 μM) and/or TAK‐779 (30 μM) were infected with stool filtrate containing GII.3 (4.3 × 10 5 GEs/well) (a) or GII.4 (1.1 × 10 7 GEs/well) (b), as shown in Figure . At 48 hpi, the cells and culture supernatants were collected, and viral RNA was quantified by RT‐qPCR. Data are presented as the mean ± SD ( n ≥ 9). Statistical significance was assessed using the Kruskal–Wallis test followed by Steel's multiple‐comparison test. Asterisks indicate statistically significant differences compared with DMSO‐treated controls (** p < 0.01, **** p < 0.0001). hpi, h postinfection.

Article Snippet: Briefly, J2 monolayers were pretreated with either ruxolitinib (5 μM; T1829, TargetMol, Boston, MA, USA), TAK‐779 (30 μM; T7499, TargetMol), both, or DMSO (control) at 37°C under 5% CO 2 for 3 h, and then inoculated with HuNoV filtrates—1.6 × 10 5 GEs/well for GII.17, 1.1 × 10 7 GEs/well for GII.4, and 4.3 × 10 5 GEs/well for GII.3—in a differentiation medium supplemented with 500 μM sodium glycochenodeoxycholate (GCDCA; Sigma‐Aldrich) with DMSO or the indicated compounds at 37°C for 1 h. The monolayers were then washed twice with complete medium without growth factors (CMGF[−]).

Techniques: Infection, Quantitative RT-PCR, Comparison

Journal: Genes to Cells

Article Title: Treatment With Ruxolitinib and TAK ‐779 Enhances GII .17 Human Norovirus Replication and Enables Serial Passaging in Human Intestinal Enteroids

doi: 10.1111/gtc.70139

Figure Lengend Snippet: Treatment with ruxolitinib and TAK‐779 enables serial passaging of GII.17, and passaged viruses exhibit higher viral RNA copies than stool‐derived viruses in HIEs. (a) J2 monolayers pretreated with ruxolitinib (5 μM) and TAK‐779 (30 μM) were infected with stool filtrate containing GII.17 (1.6 × 10 5 GEs/well) in a differentiation medium supplemented with the specified compounds and GCDCA for 3 h at 37°C. At 96 hpi, the cells were harvested, sonicated, and centrifuged. A portion of the clarified supernatants was used for RT‐qPCR quantification of viral RNA. For serial passaging, the virus was used to inoculate newly prepared J2 monolayers under the same conditions for up to five passages. Data are presented as mean ± SD ( n ≥ 5). (b) J2 monolayers were inoculated with 4.6 × 10 4 GEs per well of stool‐derived GII.17 or P5 virus stock in differentiation medium supplemented with GCDCA for 1 h at 37°C. Following infection, cells and culture supernatants were collected at 48 hpi, and viral RNA was quantified by RT‐qPCR. Data are presented as mean ± SD ( n = 4). Statistical significance was assessed using the Welch t ‐test. Asterisks indicate statistically significant differences compared with stool‐derived infection at 48 hpi (* p < 0.05). hpi, h postinfection.

Article Snippet: Briefly, J2 monolayers were pretreated with either ruxolitinib (5 μM; T1829, TargetMol, Boston, MA, USA), TAK‐779 (30 μM; T7499, TargetMol), both, or DMSO (control) at 37°C under 5% CO 2 for 3 h, and then inoculated with HuNoV filtrates—1.6 × 10 5 GEs/well for GII.17, 1.1 × 10 7 GEs/well for GII.4, and 4.3 × 10 5 GEs/well for GII.3—in a differentiation medium supplemented with 500 μM sodium glycochenodeoxycholate (GCDCA; Sigma‐Aldrich) with DMSO or the indicated compounds at 37°C for 1 h. The monolayers were then washed twice with complete medium without growth factors (CMGF[−]).

Techniques: Passaging, Derivative Assay, Infection, Sonication, Quantitative RT-PCR, Virus

Journal: Genes to Cells

Article Title: Treatment With Ruxolitinib and TAK ‐779 Enhances GII .17 Human Norovirus Replication and Enables Serial Passaging in Human Intestinal Enteroids

doi: 10.1111/gtc.70139

Figure Lengend Snippet: Detection of GII.17 VP1 in HIEs by immunofluorescence. J2 monolayers pretreated with DMSO (a) or ruxolitinib (5 μM) and TAK‐779 (30 μM) (b) were infected with mock inoculum (left panels), stool filtrate containing GII.17 (middle panels), or P4 virus stock (right panels) in the presence of DMSO or specified compounds and GCDCA. At 96 hpi, the cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X‐100. GII.17‐positive cells were detected using GII.17‐specific antiserum (green) and stained with DAPI (blue). Scale bars: 100 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; VP1, viral protein 1.

Article Snippet: Briefly, J2 monolayers were pretreated with either ruxolitinib (5 μM; T1829, TargetMol, Boston, MA, USA), TAK‐779 (30 μM; T7499, TargetMol), both, or DMSO (control) at 37°C under 5% CO 2 for 3 h, and then inoculated with HuNoV filtrates—1.6 × 10 5 GEs/well for GII.17, 1.1 × 10 7 GEs/well for GII.4, and 4.3 × 10 5 GEs/well for GII.3—in a differentiation medium supplemented with 500 μM sodium glycochenodeoxycholate (GCDCA; Sigma‐Aldrich) with DMSO or the indicated compounds at 37°C for 1 h. The monolayers were then washed twice with complete medium without growth factors (CMGF[−]).

Techniques: Immunofluorescence, Infection, Virus, Staining

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A to C ) HIEs were pretreated for 3 hours with the indicated concentrations of TAK-779 and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the absence or presence of the same concentrations of TAK-779. After washing, the cells were cultured for 48 hours at 37°C in the absence or presence of TAK-779. Viral GEs at 1 and 48 hours postinfection were quantified by RT-qPCR. Mean data compiled from two independent experiments (J2 and J8FUT2-KI HIEs) and three independent experiments (J4FUT2-KI HIEs) with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors. Significance was determined using two-way ANOVA with Tukey’s post hoc test comparing TAK-779 concentrations and individual experimental results at 48 hours postinfection. P values are shown for individual TAK-779 concentrations versus no TAK-779 treatment control (* P value <0.05, ** P < 0.01, *** P < 0.001, and ****, 0.0001). Significant differences between experiments were observed for the studies conducted in J4FUT2-KI and J8FUT2-KI HIEs ( P < 0.0001 for each).

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Control

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: Different conditions, pre- versus post-treatment (indicated) with pretreatment alone compared to post-treatment alone and 24 hours pretreatment, were compared. J4FUT2-KI HIEs were treated as indicated with 30 μM TAK-779. HIEs were then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the indicated conditions. After washing, the cells were cultured for 48 hours at 37 ° C in the presence of 30 μM TAK-779. Viral GEs at 1 and 48 hours postinfection were quantified by RT-qPCR. Mean data compiled from three independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors. Numbers above lines represent log 10 increase between no treatment versus indicated treatments. Significance was determined using two-way ANOVA with post hoc Tukey’s test comparing 48-hour replication, no TAK-779 to each condition of TAK-779 ( P value,**, < 0.001 and ****, 0.0001). hr, hours.

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Cell Culture, Quantitative RT-PCR

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated for 3 hours and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the presence of the indicated concentrations of TAK-779. After washing, the cells were cultured for the indicated times at 37°C in the presence of the indicated concentrations of TAK-779. Virus replication was evaluated by RT-qPCR at the indicated time points. ( B and C ) After 24 and 48 hours postinfection incubation, monolayers were fixed with methanol. GII.3-positive cells (green) were detected using guinea pig anti-HuNoV VLP Ab (green) and nuclei (blue) were detected by DAPI. (B) Representative images from each condition at 48 hours postinfection are shown. Scale bars denote 14 μm. (C) Numbers of HuNoV infected cells (Foci) per well from 24 and 48 hours postinfection condition were counted. Mean data compiled from two experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors using one-way ANOVA with post hoc Newman Keuls test comparing GEs per well for TAK-779 concentrations at 48- and 72-hour time points, ( P value, ****, 0.0001).

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Cell Culture, Virus, Quantitative RT-PCR, Incubation

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI and ( B ) J8FUT2-KI HIEs were pretreated for 3 hours and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) with or without 30 μM TAK-779, labeled as P1. Following infection, both cells and supernatant were collected at 96 hours postinfection, and a portion was used to quantify viral RNA by RT-qPCR. The virus was subsequently used to infect a fresh batch of HIEs for the next passage, continuing for up to five passages. dpi, days postinfection; hpi, hours postinfection.

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Labeling, Quantitative RT-PCR, Virus

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI and J8FUT2-KI HIEs were pretreated and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the presence of 30 μM TAK-779 and indicated as P1. Postinfection, the cells and supernatant were harvested at 96 hours postinfection; after processing the supernatant, an aliquot was used to quantify the viral RNA by RT-qPCR. The virus was then used to infect a new batch of HIEs for the next passage, continuing up to 10 passages. ( B ) Immunofluorescence staining of HuNoV-infected J4FUT2-KI monolayers at P5 and P2. At 48 hours postinfection, cells were fixed with methanol and stained with guinea pig anti-HuNoV VLP antibody (green), rabbit anti-NTPase (red), and mouse anti-VPg (white). Nuclei were counterstained with DAPI (blue). Representative images are shown at high (10-μm scale bar) and low (20-μm scale bar) magnification. dpi, days postinfection; hpi, hours postinfection.

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Quantitative RT-PCR, Virus, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated for 3 hours with 30 μM TAK-779 and infected with GII.3 HuNoV derived from stool [2.9 × 10 5 genome equivalents (GEs) per well] or from stock 1 (2.58 × 10 5 GEs per well), stock 2 (2.93 × 10 5 GEs per well), stock 3 (1.02 × 10 5 GEs per well), or stock 4 (2.50 × 10 5 GEs per well). Infections were performed in the presence of 30 μM TAK-779 (left) or vehicle control (right). After washing, the cells were cultured for the indicated times at 37°C in the presence (left) or absence (right) of TAK-779. Virus replication was evaluated by RT-qPCR at the indicated time points. Viral replication was assessed by RT-qPCR. The characterization of each stock was performed once with three replicates. Error bars denote SD. ( B ) Representative images of stock-infected HIEs fixed with methanol at 48 hours postinfection. GII.3-positive cells were detected using guinea pig anti-HuNoV VLP Ab (green) and nuclei (blue) were detected by DAPI. Scale bars denote 14 μm. ( C ) Electron micrograph of HuNoV particles from the supernatant of HIEs 24 hours postinfection infected with stock virus. Scale bar, 50 nm; red line, ~40 nm, expected particle diameter.

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Derivative Assay, Control, Cell Culture, Virus, Quantitative RT-PCR

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated with 30 μM TAK-779 and then infected with GII.3 (TCH-04-577/P21, 2.9 × 10 5 GE per well; TCH-25-396/P12, 2.2 × 10 5 GE per well), GII.17 (1295-44/P13, 2.8 × 10 5 GE per well; TCH-25-256/P171, 3.1 × 10 5 GE per well), G1.1 (BCM-723–100595/P1, 3.7 × 10 5 GEs per well; 51899/P1, 6.9 × 10 5 GE per well). ( B ) J4FUT2-KI HIEs were pretreated with 30 μM TAK-779 and then infected with GII.4 Sydney (BCM 16-2/P31, 3.0 × 10 5 GE per well; TCH-24-163/P16, 2.2 × 10 5 GE per well), GII.4 Den Haag (TCH-23-323/P16, 5.6 × 10 6 GE per well), GII.4 Hunter (TCH-05-797/P4, 3.35 × 10 5 GE per well) in the absence or presence of 30 μM TAK-779. After washing, the cells were cultured for 48 hours at 37°C in the absence or presence of 30 μM TAK-779. Virus replication at 1 and 48 hours postinfection was evaluated by RT-qPCR. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors Error bars denote SD. Significance was determined using paired t test comparing 48-hour replication, 0 to 30 μM TAK-779 ( P value, *, 0.01; **, 0.01; ***, 0.001; and ****, < 0.0001). hr, hours.

Article Snippet: TAK-779 was purchased from Medchem Express (catalog no. HY-13406) and reconstituted in water to make 5 mM stocks and frozen in single-use aliquots to avoid repeated freeze-thaw cycles.

Techniques: Infection, Cell Culture, Virus, Quantitative RT-PCR