Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: Viability of CHO-K1 cells after 24 h of treatment with D. superbus ( A ) and P. paradoxus ( B ) expressed as a percentage of the DMSO control (set to 100%). Data are presented as mean ± STD from three independent experiments.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques: Control

Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: The cytotoxic effects of D. superbus extracts ( A ) and P. paradoxus ( B ) extracts on CHO-K1 cells under the CBMN assay in the absence and presence of MMC. Data represents the mean values from three independent experiments.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: ( A ) The effect of different D. superbus extract treatments on the micronuclei frequency (%) in CHO-K1 cells. Cells were treated for 24 h either with DMSO at 0.3% (NC), MMC at 0.025 µg/mL or 3 different concentrations of D. superbus extract, 6.3, 12.5 and 25 µg/mL. Then cells were incubated with 3 µg/mL of cytochalasin B for another 24 h. Graphs represent data collected from 3 independent experiments. One-way ANOVA, Tukey’s multiple comparisons test using GraphPad Prism 7 software, was applied to calculate statistical significance in comparison with the NC control. (* p = 0.0155, *** p = 0.0006, **** p < 0.0001). Micronuclei frequency (%) was calculated from the following Equation: Micronuclei frequency (%) = binucleated cells with MN /binucleated cells *100. ( B ) Representative microscopic images showing micronuclei formation in binucleated CHO-K1 cells, observed with a 40× objective. CHO-K1 cell DNA was stained with Hoechst dye. Green arrows indicate the micronuclei, and the white line, labelled 50 μm, represents the scale bar in the image.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques: Incubation, Software, Comparison, Control, Staining

Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: ( A ) The effect of different D. superbus extract treatments on the micronuclei frequency (%) in CHO-K1 cells in the presence of MMC. Cells were treated for 24 h either with DMSO (NC), MMC at 0.025 µg/mL or in combnation of MMC and of D. superbus extract at 3 different concentrations 6.3, 12.5 and 25 µg/mL 24 h, then incubation with 3 µg/mL of cytochalasin B for another 24 h. Graphs represent data collected from 3 independent experiments. One-way ANOVA, Tukey’s multiple comparisons test using GraphPad Prism 7 software, was applied to calculate statistical significance in comparison with the NC control. (* p = 0.0155, *** p = 0.0006, **** p < 0.0001). Micronuclei frequency (%) was calculated from the following Equation: Micronuclei frequency (%) = binucleated cells with MN /binucleated cells *100. ( B ) Representative images illustrating micronuclei formation in binucleated CHO-K1 cells after exposure to MMC alone or in combination with the highest tested concentration of D. superbus extract.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques: Incubation, Software, Comparison, Control, Concentration Assay

Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: ( A ) The effect of different P. paradoxus extract treatments on the micronuclei frequency (%) in CHO-K1 cells. Cells were treated for 24 h with 3 different concentrations of P. paradoxus extract, 12.5, 25 and 50 µg/mL or MMC at 0.025 µg/mL, then followed by 24 h incubation with 3 µg/mL of cytochalasin B. Graphs represent data collected from 3 independent experiments. One-way ANOVA, Tukey’s multiple comparisons test using GraphPad Prism 7 software, was applied to calculate statistical significance in comparison with the NC control. (* p = 0.0435, ** p = 0.0019, *** p = 0.0007, **** p < 0.0001). ( B ) The micronuclei formation in binucleated CHO-K1 cells, observed with a 40× objective. CHO-K1 cells’ DNA was stained with Hoechst dye. Green arrows indicate the micronuclei, and the white line, labelled 50 μm, represents the scale bar in the image.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques: Incubation, Software, Comparison, Control, Staining

Journal: Scientific Reports

Article Title: Genotoxic potential of Dianthus superbus var. superbus and Petasites paradoxus (Retz.) Baumg. methanolic extracts in Chinese hamster ovary cells

doi: 10.1038/s41598-026-50267-x

Figure Lengend Snippet: ( A ) The effect of different P. paradoxus extract treatments on the micronuclei frequency (%) in CHO-K1 cells. Cells were treated for 24 h with 3 different concentrations of P. paradoxus extract, in the presence of MMC, then followed by 24 h of incubation with 3 µg/mL of cytochalasin B. Graphs represent data collected from 3 independent experiments. One-way ANOVA, Tukey’s multiple comparisons test using GraphPad Prism 7 software, was applied to calculate statistical significance in comparison with NC control. (* p = 0.0435, ** p = 0.0019, *** p = 0.0007, **** p < 0.0001). in micronuclei frequency (%) was calculated from the following Equation: ( B ) Representative microscopic images showing micronuclei formation in binucleated CHO-K1 cells, observed with a 40× objective after 24 treated with 0.025 µg/mL MMC alone or MMC + P. paradoxus extract at 25 µg/mL. CHO-K1 cell DNA was stained with Hoechst dye. Green arrows indicate the micronuclei, and the white line, labelled 50 μm, represents the scale bar in the image.

Article Snippet: The CHO-K1 cell line (603480) was purchased from CLS Cell Lines Service (GmbH, Germany).

Techniques: Incubation, Software, Comparison, Control, Staining

Journal: mBio

Article Title: Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion

doi: 10.1128/mbio.03424-25

Figure Lengend Snippet: Cell surface expression of HSV-1 and SaHV-1 gB. ( A ) CHO-K1 cells were transfected overnight with plasmids encoding WT or Flag-tagged HSV-1 or SaHV-1 gB or empty vector. Cell surface gB expression was detected by adding a polyclonal anti-HSV-1 gB antibody (R74) or a monoclonal anti-Flag antibody (F1804 M2), followed by fixative and secondary antibody. ( B ) CHO-K1 cells were transfected overnight with plasmids encoding WT gB, gB chimeras, or an empty vector. Cell surface gB expression was detected by adding an R74 PAb, followed by fixative and secondary antibody. Each bar shows the mean and standard deviation of three independent experiments. Background signals from the vector-only control were subtracted from the values. Data are normalized to the expression level of HSV-1 gB. Statistical significance was determined using Student’s t -test (ns [not significant] P ≥ 0.05; ** P < 0.01; *** P < 0.001), with HSV-1 gB as reference.

Article Snippet: Chinese hamster ovary (CHO-K1) (ATCC, USA) cells were grown in Ham’s F12 medium supplemented with 10% FBS.

Techniques: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Control

Journal: mBio

Article Title: Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion

doi: 10.1128/mbio.03424-25

Figure Lengend Snippet: Cell-cell fusion activity of HSV-1 and SaHV-1 gB chimeras with nectin-1. One set of CHO-K1 cells (effector cells) was transfected with plasmids encoding a version of gB (WT, chimera, or empty vector), gD, gH, and gL from either HSV-1 (filled bars) or SaHV-1 (open bars), and T7 polymerase. A second set of CHO-K1 cells (target cells) was transfected with plasmids carrying the luciferase gene under the control of the T7 promoter and nectin-1 receptor. Target and effector cells were cocultured for 18 hours, and luciferase activity was measured as an indication of cell-cell fusion. Data are expressed as a percentage of fusion mediated by WT HSV-1 gB for the HSV-1 gD and gH/gL samples (filled bars) or WT SaHV-1 gB for the SaHV-1 gD and gH/gL samples (open bars). Background fusion detected after transfection with the vector instead of gB was subtracted from the values. Means and standard deviations of the results of three independent experiments are shown. Bars are numbered for reference. The regions that were swapped are noted below the reciprocal chimera pairs. Statistical significance was determined using Student’s t -test (ns [not significant] P ≥ 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Chinese hamster ovary (CHO-K1) (ATCC, USA) cells were grown in Ham’s F12 medium supplemented with 10% FBS.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Control

Journal: mBio

Article Title: Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion

doi: 10.1128/mbio.03424-25

Figure Lengend Snippet: Cell-cell fusion activity of HSV-1 and SaHV-1 gB chimeras with CD155 or HVEM. Effector CHO-K1 cells were transfected with plasmids encoding a version of gB (WT, chimera, or empty vector), gD, gH, and gL from either HSV-1 (filled bars) or SaHV-1 (open bars), and T7 polymerase. Target cells were transfected with plasmids carrying the luciferase gene under the control of the T7 promoter and CD155 ( A ) or HVEM ( B ) receptor. Cells were cocultured, and luciferase activity was measured. Data are shown as a percentage of WT SaHV-1 gB (for CD155) or WT HSV-1 gB (for HVEM), following subtraction of vector-only background signal. Means and standard deviations of the results of three independent experiments are shown. Statistical significance was determined using Student’s t -test with WT SaHV-1 ( A ) or WT HSV-1 ( B ) conditions as reference (** P < 0.01; *** P < 0.001).

Article Snippet: Chinese hamster ovary (CHO-K1) (ATCC, USA) cells were grown in Ham’s F12 medium supplemented with 10% FBS.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Control

Journal: mBio

Article Title: Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion

doi: 10.1128/mbio.03424-25

Figure Lengend Snippet: Hyperfusogenic gB chimeras retain dependence on gD and gH/gL. Effector CHO-K1 cells were transfected with plasmids encoding a version of gB (WT, S1 or S2 chimera, or empty vector), gD, and/or gH and gL from either HSV-1 (filled bars) or SaHV-1 (open bars), and T7 polymerase. Target cells were transfected with plasmids carrying the luciferase gene under the control of the T7 promoter and nectin-1. Cells were cocultured, and luciferase activity was measured. Data are shown as a percentage of WT HSV-1 (filled bars) or WT SaHV-1 (open bars) fusion, following subtraction of vector-only background signal. Means and standard deviations of the results of three independent experiments are shown. Statistical significance was determined using Student’s t -test with WT SaHV-1 conditions as reference (*** P < 0.001).

Article Snippet: Chinese hamster ovary (CHO-K1) (ATCC, USA) cells were grown in Ham’s F12 medium supplemented with 10% FBS.

Techniques: Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Schemes of the workflow used in this study. (A) Main steps employed in the screening along with the number of compounds left after each step. (B) A scheme showing the detailed order of utilized techniques, especially docking to CB2 structures from PDB IDs 5ZTY and 6KPC and to the CB2 model based on MD of PDB ID 6PT0 .

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques:

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2–ligand complexes. (A–C) Binding sites with ligands (green) and amino acids (gray) important for ligand binding depicted in stick representation. PDB IDs 5ZTY , 6KPC , and 6PT0 , respectively. (D–F) 2D interaction schemes generated using Schrödinger Maestro. Additionally, we marked with gray, dashed circles the amino acids that are too far away from the ligand to create protein–ligand interactions in deposited structures but probably do so alternately, for limited periods of time in natural, nonstatic complexes.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Binding Assay, Ligand Binding Assay, Generated

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: (A) Radioligand displacement curves for two screened compounds with the lowest K i values toward human CB2—AS-5 and AS-7. WIN 55,212-2 was issued as the reference compound. Both identified CB2 ligands exhibit desired nanomolar K i and structural distinctiveness compared to the other known compounds with high affinity for CB2. (B) Inhibition of CP-55,940-stimulated [ 35 S]GTPγS at the CB2 receptor by the compounds at 10 μM. Results were expressed as mean percent of basal [ 35 S]GTPγS binding in the presence of 100 nM CP-55,940 as stimulating ligand. AM-630 served as a reference CB2 antagonist. Basal binding was set to 100% and is represented by the dotted line. Data was collected from three separate experiments and analyzed with the two-tailed t test. Statistical significance was depicted as follows: ** p < 0.01; *** p < 0.001.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Inhibition, Binding Assay, Two Tailed Test

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Best identified compound—AS-7 (green) docked to CB2 models based on PDB IDs 5ZTY (A), 6KPC (B) and 6PT0 MD-derived structure (C). Yellow dashed line, H-bond; teal dashed line, π–π interaction. (D) CB2–WIN 55,212-2 (magenta) complex from the largest 6PT0 MD cluster with AS-7 (green) docked to this model. The superposition shows, that despite the different chemotypes, the binding modes of both ligands exhibit similarities, mainly in the placement of the morpholine moieties and carbonyl oxygen atoms and to a lesser extent in the location of two AS-7 benzene rings in similar positions to WIN 55,212-2 central tricyclic moiety and naphthyl group.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay, Binding Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: AS-5 (green) docked to CB2 models based on PDB IDs 5ZTY (A) and 6PT0 MD-derived structure (B). Yellow dashed line, H-bond; teal dashed line, π–π interaction.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2 Structures Deposited in PDB a

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Selected Results of the K i Determination with [ 3 H]CP-55,940 Displacement Assay

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Docking and MM–GBSA Results for the Four Most Potent Compounds from the In Vitro Assay and Three Already Known CB2 Ligands for Comparison

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: In Vitro