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: 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

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Western blot showing the expression of GPR44 and β-actin in purified 1° WT and Gpr44 −/− AML cells. GPR44 antibody detects glycosylated (63 kDa) and unglycosylated (33 and 50 kDa) and forms. (B) CD45.2- and Ai14 TdTomato -recipient mice were maintained on Se-S diet for 4 weeks before transplantation until the endpoint. 2° transplantation was done retro-orbitally with 1° CD45.1 WT or CD45.2 Gpr44 −/− AML donor cells to CD45.2= and Ai14 TdTomato -recipient mice, respectively; 3 weeks later, mice were euthanized; blood, bone marrow, and spleen were sampled (n = 8 in each group). (C) CBC analysis of Se-S AML mice in (B). (D and E) Counts of AML cells in the Lin − population in the bone marrow (D) and spleen (E) of Se-S AML mice in (B). (F and G) Counts of LICs (WT: CD45.1 + Lin − Sca-1 − c-Kit + , see also ; Gpr44 −/− : RFP − Lin − Sca-1 − c-Kit + , see also ) in the bone marrow (F) and spleen (G) of Se-S AML mice in (B). (H) Survival curve of recipient mice with competitive 2° transplantation of WT (4 × 10 5 ), WT + Gpr44 −/− (2 × 10 5 : 2 × 10 5 ), or Gpr44 −/− (4 × 10 5 ) AML donor cells (n = 6–9 in each group). (I) Progression of WBCs in the peripheral blood of recipient mice secondarily transplanted with WT or Gpr44 −/− AML donor cells (n = 8–9 in each group). (J) Purified 1 WT and Gpr44 −/− AML cells were plated in methylcellulose medium (2,500 cells/well, 4 replicates). CFUs were counted on day 8. (K) Representative image of colony growth from purified 1 WT and Gpr44 −/− AML cells. Scale bar, 100 μm. (L) Comparison of GPR44 expression in blood cancers compared with normal subjects. (M) Comparison of GPR44 expression in AML FAB subtypes including M0, M1, M2, M3, M4, M4Eo, M5, M6, and M7. (L and M) Data were generated from the ONCOMINE database. (L) Each point represents a comparison of the study between the cancer population and normal population. (M) Each point represents a comparison of the study of one FAB subtype with the other subtypes. Plots were generated using the −log10 (p value) and the fold change in expression. p values were obtained by t test of the mean values. An absolute fold change of 1.5 (red line) or higher is considered significant. Data shown are mean ± SEM per group; *p < 0.05, **p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Western Blot, Expressing, Purification, Transplantation Assay, Generated

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Scheme for sequencing analysis. Bone marrow cells were isolated from mice secondarily transplanted with 1° WT or Gpr44 −/− AML donor cells and LICs were flow cytometrically sorted following RBC lysis, Lin − selection, and Sca-1 and c-Kit staining. LICs (4 × 10 5 ) were used for RNA sequencing. Differential gene expression analysis, IPA, and GSEA were performed to compare WT and Gpr44 −/− LICs (n = 3 in each group). (B) Heatmap for top 100 most differentially regulated genes in WT and Gpr44 −/− LICs. (C) Volcano plot for differential gene expression analysis between WT and Gpr44 −/− LICs (D) Heatmap of indicated pathways in WT and Gpr44 −/− LICs as analyzed by IPA. Z scores were plotted. (E) Expression of Kras assessed by qPCR analysis in WT or Gpr44 −/− AML cells isolated from bone marrow (left) and spleen (right) of 2° AML-recipient mice. Data were normalized to WT AML cells and 18S rRNA expression (n = 4–8 biological replicates). (F) Western blot showing the expression of KRAS in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice. (G) Western blot showing the expression of MAPK signaling pathway components including P-C-RAF, RAF-1, P-MEK3/6, MEK1/2, P-ERK, ERK1/2, P-C-JUN,and β-actin in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice (n = 5–8 biological replicates in each group). (H) Western blot showing the expression of P53 in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice. (F and H) Densitometry was done by normalizing to WT AML cells and relative to β-actin (n = 3–5 biological replicates in each group). Data shown are mean ± SEM per group; *p < 0.05, **p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Sequencing, Isolation, Lysis, Selection, Staining, RNA Sequencing Assay, Expressing, Western Blot

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Scheme showing the treatment of BAY293 and CCK8 assay on unpurified Gpr44 −/− AML cells. Frequency of AML cells was above 95%. (B) Comparison of the viability of unpurified Gpr44 −/− AML cells treated with BAY293 (0, 5, 10, and 15 μM, n = 3) for 24 or 48 h. Data were normalized to 0 μM (24 h) treatment. **p < 0.01, comparison between concentrations in the 24 and 48 h time points analyzed by Student’s t test; ## p < 0.01, comparison between time points analyzed by two-way ANOVA followed by appropriate post hoc test (Bonferroni correction). (C) Scheme showing the treatment of BAY293 and live/dead cell measurement on purified Gpr44 −/− AML cells. CD45.2 Gpr44 −/− AML cells were transplanted into CD45.1 recipients; purification of Gpr44 −/− AML cells isolated from spleen was done by CD45.1-positive magnet selection kit. (D and E) Frequency (D) and count (E) of live cells in purified Gpr44 −/− AML cells treated with 15 μM BAY293 for 48 h (n = 3). (F) Western blot showing the expression of P-MEK3/6 and P-ERK in purified 1° Gpr44 −/− AML cells transduced with pLV hU6-sgRNA KRAS hUbC-dCas9-KRAB-T2a-GFP virus. (G) Western blot showing the expression of KRAS in purified 2° Gpr44 −/− AML cells transduced with pLV hU6-sgRNA KRAS hUbC-dCas9-KRAB-T2a-GFP virus. Densitometry was done by normalizing to control group and relative to β-actin (n = 3–4 in each group). (H) Survival curve of AML mice tertiarily transplanted with purified KRAS KD Gpr44 −/− AML cells (n = 7). (I and J) Survival analysis of mice tertiarily transplanted PD98059-treated Gpr44 −/− AML cells. Gpr44 −/− AML cells were cultured ex vivo with or without 100 μM PD98059 for 24 h and then retro-orbitally transplanted into mice (n = 6 per group). Survival was followed up for 60 days post transplantation. (K) Western blot showing the expression of P-ERK and P53 in Gpr44 −/− AML cells treated with PD98059 (0, 10, 50, and 100 μM) for 24, 48, and 72 h. (L and M) Representative image of colony growth from purified 2° Gpr44 −/− AML cells (2,500 cells/well, n = 4 for biological replicate, n = 2 for technical replicate) treated with 100 μM PD98059 and 15 μM BAY293 in methylcellulose medium. Scale bar, 100 μm. (N) Counts of CFUs from purified 2° Gpr44 −/− AML cells treated with 100 μM PD98059 and 15 μM BAY293 in methylcellulose medium. CFUs were counted on day 8. Data shown are mean ± SEM per group; *p < 0.05, ** p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: CCK-8 Assay, Purification, Isolation, Selection, Western Blot, Expressing, Transduction, Cell Culture, Ex Vivo, Transplantation Assay

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A–D) Expression of genes including PI3K ( Pik3ca , Pik3cb , Pik3cd ) (A), AKT ( Akt1 , Akt2 ) (B), PKC ( Prkca , Prkcb , Prkcg ) (C), and PKA ( Prkacg ) (D) assessed by qPCR analysis in WT or Gpr44 −/− AML cells isolated from spleens of 2 AML-recipient mice. Data were normalized to WT AML cells and Gapdh expression (n = 8–10 biological replicates in each group). (E) Western blot showing the expression of Phospho-PI3K, PI3K, Phospho-mTOR, mTOR, Phospho-AKT, AKT, Phospho-P70-S6K, P70-S6K, and β-actin in purified 1° WT and Gpr44 −/− AML cells. (F) Western blot showing the expression of Phospho-PTEN, PTEN, Phospho-4EBP1, 4EBP1, and β-actin in WT or Gpr44 −/− AML cells isolated from spleens of 2° AML-recipient mice (n = 5 biological replicates in each group). (G) Viability of purified 1 Gpr44 −/− AML cells treated with 150 μM ARN509, 10 μM LY294002, 10 nM Wortmannin, 10 nM Sapanisertib, 250 nM Torin1 for 24 h. Data were normalized and compared with vehicle treatment. (H and I) Flow cytometric analysis of Phospho-P70-S6K (H) and Phospho-4E-BP1 (I) in CD45.1 + 1° Gpr44 −/− AML cells treated with 10 μM LY294002, 10 nM Wortmannin, 10 nM Sapanisertib, 250 nM Torin1 for 24 h. MFI was summarized (n = 4). (J) Counts of CFUs of purified 1° Gpr44 −/− AML cells treated with 10 μM LY294002, 10 nM Sapanisertib, and 2.5 μM LY2584702 in methylcellulose medium (2,500 cells/well, n = 3 for technical replicate) CFUs were counted on day 8. (K) Representative image of colony growth in (J). Scale bar, 100 μm. Data shown are mean ± SEM per group; *p < 0.05, ** p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Expressing, Isolation, Western Blot, Purification

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Concentration Assay, Over Expression, Protein Extraction, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Frontiers in Molecular Biosciences

Article Title: Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells

doi: 10.3389/fmolb.2023.1119157

Figure Lengend Snippet: Time course of FA change caused by CELT-419 binding to D 3 receptor on the BBV particles. The reaction was initiated by the addition of 1 μL D 3 receptor displaying BBVs (C D3R = 0.7 nM) to 0.5 nM CELT-419 in the absence (blue circles) or presence (black squares) of 50 μM Spiperone, or solution without BBVs (orange diamonds). After 90 min (indicated with an arrow), the measurement was paused, and dissociation was initiated by the addition of 333 μM Spiperone (open light blue circles). An equivalent volume of assay buffer was added to association controls (filled dark blue circles). A representative experiment of at least three independent experiments is shown.

Article Snippet: For D 3 receptors in each well was incubated 2 μg of membranes from D 3 cell line (Perkin Elmer, ES-173M400UA, protein concentration = 1000 μg/ml), 1 nM [ 3 H]-Spiperone (68 Ci/mmol, 1 mCi/ml, Perkin Elmer NET1187001MC) with compounds studied or a standard.

Techniques: Binding Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells

doi: 10.3389/fmolb.2023.1119157

Figure Lengend Snippet: Binding curves of CELT-419 binding to D 3 receptors in BBVs. FA of 3 nM (orange squares) or 0.5 nM (blue circles) CELT-419 were measured after 2 h incubation with different amounts of D 3 receptor displaying BBVs. Non-specific binding (NS, open symbols) was determined in the presence and total binding (T, filled symbols) in absence of 50 μM Spiperone. The displayed fit, the concentration of D 3 receptor binding sites and the K d values were calculated post hoc from the results of these experiments using the model described in . K d ± SEM is the weighted average of three independent experiments performed in duplicates. Representative experiment performed in duplicates is shown, with both duplicates displayed.

Article Snippet: For D 3 receptors in each well was incubated 2 μg of membranes from D 3 cell line (Perkin Elmer, ES-173M400UA, protein concentration = 1000 μg/ml), 1 nM [ 3 H]-Spiperone (68 Ci/mmol, 1 mCi/ml, Perkin Elmer NET1187001MC) with compounds studied or a standard.

Techniques: Binding Assay, Incubation, Concentration Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells

doi: 10.3389/fmolb.2023.1119157

Figure Lengend Snippet: Displacement of CELT-419 binding to D 3 receptor by different dopaminergic ligands. Change in FA level was measured after incubation of 0.5 nM CELT-419, 1 μL BBV/well (C D3R = 0.7 nM), and different concentrations of corresponding dopaminergic ligands for 180 min. The FA values were normalized by taking the average FA value of C = 0 points as 100% and the average FA value of the highest used concentration points as 0%. Data of a representative experiment from at least three independent experiments, performed in duplicates, is shown with both replicates displayed. pK i values are calculated as described in , and presented as mean ± SD of n independent experiments.

Article Snippet: For D 3 receptors in each well was incubated 2 μg of membranes from D 3 cell line (Perkin Elmer, ES-173M400UA, protein concentration = 1000 μg/ml), 1 nM [ 3 H]-Spiperone (68 Ci/mmol, 1 mCi/ml, Perkin Elmer NET1187001MC) with compounds studied or a standard.

Techniques: Binding Assay, Incubation, Concentration Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells

doi: 10.3389/fmolb.2023.1119157

Figure Lengend Snippet: Fluorescence and bright-field images of total (left panels) and non-specific binding (central panels) of CELT-419 to live HEK293-D3R cells and to SKOV3 cells without D 3 receptors (right panels). HEK293-D3R cells in DMEM medium with added 9% FBS and antibiotic antimycotic solution were incubated with 2 nM CELT-419 in the absence (total binding) or presence (non-specific binding) of 10 μM Spiperone for 2 h in 5% CO 2 and at 37 °C. The number of seeded cells per well was 20,000. For negative control, SKOV3 cells without D 3 receptors seeded at 30,000 cells per well were used with 2 nM CELT-419. The contrast of fluorescence and bright-field images was enhanced for presentation purposes only, the same lookup table was used for all images of the same imaging mode. The scale bar corresponds to 10 μm.

Article Snippet: For D 3 receptors in each well was incubated 2 μg of membranes from D 3 cell line (Perkin Elmer, ES-173M400UA, protein concentration = 1000 μg/ml), 1 nM [ 3 H]-Spiperone (68 Ci/mmol, 1 mCi/ml, Perkin Elmer NET1187001MC) with compounds studied or a standard.

Techniques: Fluorescence, Binding Assay, Incubation, Negative Control, Imaging

Journal: Frontiers in Molecular Biosciences

Article Title: Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells

doi: 10.3389/fmolb.2023.1119157

Figure Lengend Snippet: Saturation binding of CELT-419 binding to D 3 receptors on live HEK293-D3R cells. The HEK293-D3R cells (20,000 cells per well) were incubated with CELT-419 (twofold serial dilutions up to 4 nM) for 4.3 h. Non-specific binding (orange triangles) was measured in the presence of 10 μM Spiperone. The background-corrected fluorescence intensities of cells were determined with the cell detection and image quantification software as described in Materials and methods and are presented as individual replicates from a representative experiment of three independent experiments performed in duplicates. Every point corresponds to the difference between the average pixel intensity of a cell and the average pixel intensity of the background. The average pixel intensities of each point were calculated from four images from different fields of view obtained from a single well. The calculated K d value is given as mean ± SEM of three independent experiments.

Article Snippet: For D 3 receptors in each well was incubated 2 μg of membranes from D 3 cell line (Perkin Elmer, ES-173M400UA, protein concentration = 1000 μg/ml), 1 nM [ 3 H]-Spiperone (68 Ci/mmol, 1 mCi/ml, Perkin Elmer NET1187001MC) with compounds studied or a standard.

Techniques: Binding Assay, Incubation, Fluorescence, Software