Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: Targeting the CCL5–CCR5 axis attenuates neutrophil hyperactivation. (A) The effect of CCR5 inhibitors on LPS/CCL5-induced NETs formation by using Sytox Green staining. Primary neutrophils isolated from WT and PP4 myel−KO mice were pre-treated with TAK-779 (10 nM, 1hr) or UK-427857 (10 nM, 1hr), and further stimulated with either LPS (1 mg/ml) or CCL5 (50 ng/ml) for 24 h. (B and C) The production of mitoROS (B) and elastase activity (C) by neutrophils as in A, was quantified. (D) Differentiated HL-60 (dHL60) cells treated with siNT or siCCR5 were analyzed by IB using the indicated antibodies. (E-H) NETs formation (E), mitoROS (F), elastase activity (G), and immunofluorescence images of myeloperoxidase (MPO, green) and DAPI (blue) co-localization (H) in dHL60 cells transfected with siNT or siCCR5, followed by LPS stimulation with or without CCL5. All results are presented as the means ± SE.* p < 0.05; ** p < 0.01 (by a one-way ANOVA). Scale bar = 50 mm. (n = 3). (I) Schematic representation of the central role of PP4 in neutrophil recruitment and activation.

Article Snippet: TAK-779, UK-427857 (CCR5 inhibitors), PD98059 (an ERK1/2 inhibitor), SB203580(a p38 MAPK inhibitor), U0126 (MEK1 and MEK2 inhibitors), N-acetyl- l -cysteine (NAC), gefitinib (EGFR tyrosine kinase inhibitor) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Isolation, Activity Assay, Immunofluorescence, Transfection, Activation Assay

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

Journal: Cell Death & Disease

Article Title: FGFR4 promotes CAF activation through the CXCL10-CXCR3 axis in colon cancer

doi: 10.1038/s41419-025-07588-y

Figure Lengend Snippet: A Collagen gel contraction assay results show the effect of recombinant CXCL10 protein on the contractility of NIH/3T3 cells (n = 6/group). Representative gel images are shown together with the quantification of collagen gel contraction. B Migration and invasion assay results show the effect of recombinant CXCL10 protein on the migration and invasion properties of NIH/3T3 cells. The effect of CXCL10 was compared to that of TGFβ, which induces CAF activation. C The effect of recombinant CXCL10 protein (ng) on CAF marker gene levels in NIH/3T3 cells was determined using western blotting and RT-qPCR analyses. D Effect of Cxcl10 knockdown on CAF marker gene expression. NIH/3T3 cells were incubated for 24 h with conditioned media (CM) obtained from siNC- or siCXCL10-transfected CT-26/FGFR4 cells. Western blotting and RT-qPCR analyses of CAF marker expression in NIH/3T3 cells. E Effect of CXCL10 neutralizing antibody on CAF marker gene expression. NIH/3T3 cells were incubated in CM obtained from CT-26/FGFR4 cells in the presence of either normal anti-IgG or anti-CXCL10 neutralizing antibodies (concentrations shown in μg/mL) for 24 h. F Effect of AMG487, a pharmacological CXCR3 inhibitor, on gene expression of CAF markers. The CM of CT-26/FGFR4 cells, alone or in combination with the CXCR3 inhibitor, was used to treat the NIH/3T3 cells for 24 h. G Invasion assay results depicting the effect of the CXCR3 inhibitor AMG487 (2 μM) on the invasion capabilities of CT-26 primary tumor-derived CAFs. H Invasion assay results show the effects of various CXCR3 inhibitors on the invasive properties of CT26/FGFR4 tumor-derived CAFs. The CXCR3 inhibitors used (at 2 μM) were: TAK779, NBI74330, and SCH546738. Data are presented as mean ± SD. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 using Student’s t -test and one-way ANOVA). NT no treatment, AMG AMG487, TAK TAK779, NBI NBI74330, SCH SCH54673 .

Article Snippet: The following inhibitors were used: BLU9931 (Merck, Kenilworth, NJ, USA; 538776), AMG487 (MedChemExpress, Monmouth Junction, NJ, USA; HY-15319), TAK779 (MedChemExpress; HY-13406), NBI74330 (MedChemExpress; HY-15320), and SCH546738 (MedChemExpress; HY-10017).

Techniques: Collagen Gel Contraction Assay, Recombinant, Migration, Invasion Assay, Activation Assay, Marker, Western Blot, Quantitative RT-PCR, Knockdown, Gene Expression, Incubation, Transfection, Expressing, Derivative Assay