Journal: STAR Protocols

Article Title: Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay

doi: 10.1016/j.xpro.2023.102461

Figure Lengend Snippet:

Article Snippet: HaloTag mouse monoclonal antibody , Promega , cat# G9211; RRID:AB_2688011.

Techniques: Recombinant, Western Blot, Staining, Construct, Software, Cell Counting, Transferring

Journal: Virus Research

Article Title: Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

doi: 10.1016/j.virusres.2023.199176

Figure Lengend Snippet: Construction and characterization of the Δorf2.4 replicon and Δorf2 replicon. (A) Schema of the two new replicons. The Δorf2 replicon lacks only S (the brown box), while the Δorf2.4 replicon lacks S and E (the green box). (B) Electrophoretic image of the pCC2Fos-Δorf2.4 replicon and pCC2Fos-Δorf2 replicon digested with ZraI. In each of the migrating images, the larger bands are similar to each replicon and the smaller bands to pCC2Fos. The theoretical band sizes of the replicons are 25,927 and 26,179 bp for the Δorf2.4 replicon and Δorf2 replicon, respectively. (C) HiBiT signal kinetics of all three replicons. Huh-7 cells were electroporated with each replicon and the HiBiT signal was measured at the indicated time points. The significance of differences was assessed by a two-way ANOVA, considering p <0.05 as significant (**= p <0.01, ***= p <0.001, ****= p <0.0001). ns means Not Significant. (D) Immunofluorescent assay for viral protein detection. At 48 hpt, NSP8 was stained using the mouse anti-SARS-CoV-2 NSP8 antibody and goat anti-mouse IgG antibody conjugated with Alexa Fluor568 (red-colored cells), and the membrane was stained using the rabbit anti-SARS-CoV-2 membrane antibody and goat anti-rabbit IgG antibody conjugated with the Alexa Fluor 488 antibody (green-colored cells). The nucleus was stained by DAPI. (E) HiBiT luminescence in the supernatant of Δorf2 replicon-transfected cells. Huh-7 cells were electroporated with the Δorf2 replicon and HiBiT luminescence in its supernatant was measured at the indicated timepoints. For panels C and E, the averages and standard errors of at least 2 independent experiments with triplicate samples are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse anti-HiBiT tag mAb (30E5, Promega) and goat anti-mouse IgG H&L conjugated with Alexa Fluor 568 (Abcam) were used for primary and secondary antibody reactions, respectively.

Techniques: Staining, Transfection

Journal: Virus Research

Article Title: Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

doi: 10.1016/j.virusres.2023.199176

Figure Lengend Snippet: Puromycin treatment. (A) HiBiT kinetics of two puro replicons without puromycin pressure. Huh-7 cells were electroporated with the Δorf2–8 puro replicon or Δorf2.4-puro replicon, and the HiBiT signal was measured at the indicated timepoints. (B) Kinetics of replicon RNA copy numbers. The total RNA of Huh-7 cells electroporated with the puro replicon was extracted and the expression of the N gene was quantified by qRT-PCR. The graph shows the relative replicon (N) RNA copy number normalized by the copy number at 2 hpt. (C) HiBiT luminescence under the puromycin treatment. Huh-7 cells were electroporated by the Δorf2.4-puro replicon and treated with various concentrations of puromycin at 24 hpt. The medium was changed again at 72 hpt. The signal at 24 hpt is the same between samples considering the baseline. (D) Immunofluorescent assay (IFA) for the detection of HiBiT. The expression of HiBiT was detected with the anti-HiBiT tag mAb and goat anti-mouse IgG antibody conjugated with Alexa Fluor 568 (red-colored cells). The nucleus was stained by DAPI. For panels A, B and C, the averages and standard errors of at least 2 independent experiments with triplicate samples are shown. The significance of differences was assessed by a two-way ANOVA considering p <0.05 as significant (**= p <0.01, ***= p <0.001, ****= p <0.0001). ns means Not Significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse anti-HiBiT tag mAb (30E5, Promega) and goat anti-mouse IgG H&L conjugated with Alexa Fluor 568 (Abcam) were used for primary and secondary antibody reactions, respectively.

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Genome editing of human E-selectin to attach the 11 amino acid HiBiT sequence to the N terminus (A) Schematic of the position of HiBiT on the N terminus of E-selectin and its re-complementation with LgBiT. The E-selectin structure shown is the extended structure of the N-terminal regions of E-selectin (PDB: 4C16 ) showing the binding site for glycomimetic 1/sLe x and the N-terminal Lectin (Lec), EGF-like and first two SCR domains. (B) General genome-editing strategy showing the PAM sequence adjacent to the signal sequence of E-selectin and the insertion of HiBiT and a GSSG linker immediately following the signal sequence in the HiBiT-tagged E-selectin sequence. The editing disrupts the PAM sequence thus preventing any further modification by Cas9. (C) Agarose gel (3%) of a PCR amplified 370 bp fragment of the N-terminal region of E-selectin in wild-type HUVECs and HiBiT-edited TERT2-HUVEC clone C8. Successful homozygous edit of HiBiT TERT2-HUVEC clone C8 was demonstrated by the appearance of a single band representing the 45 bp addition (HiBiT + GSSG linker) in length of the PCR fragment compared to unedited HUVECs. A 100 bp ladder is also shown for comparison. (D) Bioluminescence imaging of HiBiT TERT2-HUVEC clone C8 cells expressing HiBiT-E-selectin under endogenous promotion. Cells were treated with 1 nM TNFα for 6 h and then incubated with purified LgBiT protein (50 nM in 0.1% BSA HBSS) for 20 min (37°C). Furimazine was then added (1:400 in 0.1% BSA HBSS) (5 min, RT) and 512 x 512 images were captured using bioluminescence filters (exposure 20 s, gain 200) on the Olympus LuminoView 200. Scale bar represents 50 μm. The image shown is representative of 5 similar experiments.

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Sequencing, Binding Assay, Modification, Agarose Gel Electrophoresis, Amplification, Imaging, Expressing, Incubation, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: TNFα-stimulated nanoluciferase luminescence in HiBiT-E-selectin-edited HUVECs and HiBiT-edited TERT2-HUVECs (A, C, and E) Increase in luminescence (relative light units) under basal conditions and in response to 6 h stimulation with 1 nM TNFα in HiBiT genome-edited HUVECs (A), mixed populations of HiBiT-edited TERT2-HUVECs (C) and HiBiT TERT2 clone C8 HUVECs (E). (B, D, and F) Concentration-response curves for TNFα-stimulated luminescence in HiBiT HUVECs (B), HiBiT TERT2-HUVEC mixed populations (D), and HiBiT TERT2 clone C8 HUVECs (F). NanoBiT complementation was achieved by addition of purified LgBiT (50 nM) and furimazine substrate (1:400), and the plates were read using an BMG PHERAstar FS plate reader (450 nm, 30 nm bandpass). Values are mean ± S.E.M of 5 replicate experiments each performed in triplicate. ∗p < 0.05 compared to vehicle control (paired t test).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Concentration Assay, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Cytokine-stimulated nanoluciferase luminescence in clonal HiBiT-edited E-selectin TERT2-HUVECs (A, C, and E) Increase in luminescence (relative light units) under basal conditions and in response to 6 h stimulation with 50 nM LPS (A), 500 nM IL-1α (C), and 500 nM IL-1β (E) in clone C8 HiBiT genome-edited TERT2-HUVECs. (B, D, and F) Concentration-response curves for cytokine-stimulated luminescence in clone C8 HiBiT TERT2-HUVECs in response to LPS (B), IL-1α (D), and IL-1β (F). NanoBiT complementation was achieved by addition of purified LgBiT (50 nM) and furimazine substrate (1:400), and the plates were read using a BMG PHERAstar FS plate reader (450 nm, 30 nm bandpass). Values are mean ± S.E.M of 5 replicate experiments each performed in triplicate. ∗p < 0.05 or ∗∗p < 0.01 compared to vehicle control (paired t test).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Concentration Assay, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Label-free angiogenesis assays to confirm the phenotype of CRISPR-Cas9 gene-edited TERT2-HUVECs measured using the PhaseFocus Livecyte Wild-type HUVECs, wild-type TERT2-HUVECs, and CRISPR/Cas9 gene-edited TERT2-HUVECs expressing HiBiT E-selectin (clone C8) were seeded at 20,000 cells/well onto black, flat bottomed plates pre-coated with 60 μL/well Geltrex. Cells were immediately placed within the humidified LivecyteCell Imaging chamber (Phasefocus) maintained at 37°C 5% CO 2 in air and left to equilibrate for 20 min. (A) A single 1 mm 2 region per well was imaged with a 10× objective every hour for 10 h. Integrated angiogenesis network analysis was performed using PhaseFocus Cell Analysis Toolbox version 3.8.1 on each well over the total length of the assay. Images shown are for the 10 h time point. (B) Example of the analysis with individual cells indicated by multicolored shapes and tube networks shown in yellow. (C) The total length of the network (μm) was calculated on a per cell basis at the 10 h time point. Data are expressed as the mean ± S.E.M of 4 independent experiments (4–5 wells analyzed per cell line, per experiment).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: CRISPR, Expressing, Imaging

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Effect of histamine and VEGF 165 a on HiBiT E-selectin expression in gene-edited TERT2-HUVEC clone C8 (A and B) Increase in luminescence under basal conditions and in response to 8 h stimulation with (A) 100 nM histamine or (B) a combination of 100 nM histamine and 1 nM TNFα in HiBiT genome-edited clone C8 TERT2-HUVECs. (C and D) Increase in luminescence under basal conditions and in response to 8 h stimulation with 100 nM VEGF 165 a (C) or (D) a combination of 100 nM VEGF 165 a and 1 nM TNFα in HiBiT genome-edited clone C8 TERT2-HUVECs. Values represent mean ± S.E.M of the five independent experiments, each performed in triplicate. ∗p < 0.05 compared to vehicle control (paired t test). ∗∗p < 0.01 compared to vehicle control or #p < 0.05 compared to TNFα alone (One-way ANOVA).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Kinetic profile of TNFα-stimulated luminescence in clonal HiBiT-edited E-selectin TERT2-HUVECs To monitor the real-time kinetics of TNFα-induced expression of E-selectin, experiments were undertaken with the homozygous TERT2-HUVEC HiBiT E-selectin clone C8. As these kinetic experiments were performed over 15 h, the long acting caged nanoluciferase substrate endurazine was used. (A) Time course of HiBiT E-selectin expression over 15 h in the continued presence of 50 nM LgBiT and endurazine. Values represent mean ± S.E.M from five independent experiments. (B) Incubation with 1 nM TNFα in the continued presence of 50 nM LgBiT and endurazine yielded a significant increase in luminescence over 8 h (∗∗∗p < 0.001; paired t test of 5 independent experiments). (C) To investigate potential depletion of LgBiT through the experiment, experiments were either performed in the continuous presence of 50 nM LgBiT incubation or following a 20 min addition of LgBiT (50 nM) at the end of the 15 h incubation with 1 nM TNFα. There was no significant difference between the luminescence values obtained. (D) Time course of HiBiT E-selectin expression in response to increasing concentrations of TNFα. Values represent mean ± S.E.M from six (or five, 0.25 nM) independent experiments. (E) Peak responses from (D) expressed as a percentage of the response to 1 nM TNFα. (F) Log peak responses to increasing concentrations of TNFα. Data taken from (E). ∗∗∗∗p < 0.0001 (one-way ANOVA with Dunnett multiple comparison test). Symbols show the individual means in 5 or 6 independent experiments.

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing, Incubation

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Produced, Recombinant, Transfection, Modification, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Blocking Assay, Staining, Purification, Electroporation, Luciferase, Plasmid Preparation, Software

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: TNFα-stimulated increase in E-selectin expression in un-transfected HUVECs (A) Confocal images of TNFα-stimulated E-selectin expression in wild-type (WT) HUVECs and immortalized TERT2-HUVECs. Cells were stimulated with 1 nM TNFα for 6 (WT HUVECs) or 8 h (TERT2-HUVECs). H33342-stained nuclei are shown in blue and Alexa Fluor 488-labeled E-selectin in green. Scale bar represents 50 μm. (B–D) Quantification of E-selectin expression in WT HUVECs using an alkaline phosphatase secondary antibody and p -nitrophenol phosphate substrate. Values show mean ± S.E.M. of the optical densities obtained in five separate experiments in response to 1 nM TNFα. (C and D) Concentration-response curve (C) and time course (D) for TNFα-stimulated E-selectin expression in WT HUVECs. Values are mean ± S.E.M. of five separate experiments each performed in triplicate. In (C), values have been normalized to the response obtained with 1 nM TNFα. In (D), 1 nM TNFα was used, and values have been normalized to the response obtained at 8 h. ∗∗p < 0.001 compared to vehicle control (paired t test).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing, Transfection, Staining, Labeling, Concentration Assay

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Genome editing of human E-selectin to attach the 11 amino acid HiBiT sequence to the N terminus (A) Schematic of the position of HiBiT on the N terminus of E-selectin and its re-complementation with LgBiT. The E-selectin structure shown is the extended structure of the N-terminal regions of E-selectin (PDB: 4C16 ) showing the binding site for glycomimetic 1/sLe x and the N-terminal Lectin (Lec), EGF-like and first two SCR domains. (B) General genome-editing strategy showing the PAM sequence adjacent to the signal sequence of E-selectin and the insertion of HiBiT and a GSSG linker immediately following the signal sequence in the HiBiT-tagged E-selectin sequence. The editing disrupts the PAM sequence thus preventing any further modification by Cas9. (C) Agarose gel (3%) of a PCR amplified 370 bp fragment of the N-terminal region of E-selectin in wild-type HUVECs and HiBiT-edited TERT2-HUVEC clone C8. Successful homozygous edit of HiBiT TERT2-HUVEC clone C8 was demonstrated by the appearance of a single band representing the 45 bp addition (HiBiT + GSSG linker) in length of the PCR fragment compared to unedited HUVECs. A 100 bp ladder is also shown for comparison. (D) Bioluminescence imaging of HiBiT TERT2-HUVEC clone C8 cells expressing HiBiT-E-selectin under endogenous promotion. Cells were treated with 1 nM TNFα for 6 h and then incubated with purified LgBiT protein (50 nM in 0.1% BSA HBSS) for 20 min (37°C). Furimazine was then added (1:400 in 0.1% BSA HBSS) (5 min, RT) and 512 x 512 images were captured using bioluminescence filters (exposure 20 s, gain 200) on the Olympus LuminoView 200. Scale bar represents 50 μm. The image shown is representative of 5 similar experiments.

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Sequencing, Binding Assay, Modification, Agarose Gel Electrophoresis, Amplification, Imaging, Expressing, Incubation, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: TNFα-stimulated nanoluciferase luminescence in HiBiT-E-selectin-edited HUVECs and HiBiT-edited TERT2-HUVECs (A, C, and E) Increase in luminescence (relative light units) under basal conditions and in response to 6 h stimulation with 1 nM TNFα in HiBiT genome-edited HUVECs (A), mixed populations of HiBiT-edited TERT2-HUVECs (C) and HiBiT TERT2 clone C8 HUVECs (E). (B, D, and F) Concentration-response curves for TNFα-stimulated luminescence in HiBiT HUVECs (B), HiBiT TERT2-HUVEC mixed populations (D), and HiBiT TERT2 clone C8 HUVECs (F). NanoBiT complementation was achieved by addition of purified LgBiT (50 nM) and furimazine substrate (1:400), and the plates were read using an BMG PHERAstar FS plate reader (450 nm, 30 nm bandpass). Values are mean ± S.E.M of 5 replicate experiments each performed in triplicate. ∗p < 0.05 compared to vehicle control (paired t test).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Concentration Assay, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Cytokine-stimulated nanoluciferase luminescence in clonal HiBiT-edited E-selectin TERT2-HUVECs (A, C, and E) Increase in luminescence (relative light units) under basal conditions and in response to 6 h stimulation with 50 nM LPS (A), 500 nM IL-1α (C), and 500 nM IL-1β (E) in clone C8 HiBiT genome-edited TERT2-HUVECs. (B, D, and F) Concentration-response curves for cytokine-stimulated luminescence in clone C8 HiBiT TERT2-HUVECs in response to LPS (B), IL-1α (D), and IL-1β (F). NanoBiT complementation was achieved by addition of purified LgBiT (50 nM) and furimazine substrate (1:400), and the plates were read using a BMG PHERAstar FS plate reader (450 nm, 30 nm bandpass). Values are mean ± S.E.M of 5 replicate experiments each performed in triplicate. ∗p < 0.05 or ∗∗p < 0.01 compared to vehicle control (paired t test).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Concentration Assay, Purification

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: EC 50 values for cytokine-stimulated HiBiT E-selectin expression in gene-edited wild-type and TERT2-HUVECs

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Label-free angiogenesis assays to confirm the phenotype of CRISPR-Cas9 gene-edited TERT2-HUVECs measured using the PhaseFocus Livecyte Wild-type HUVECs, wild-type TERT2-HUVECs, and CRISPR/Cas9 gene-edited TERT2-HUVECs expressing HiBiT E-selectin (clone C8) were seeded at 20,000 cells/well onto black, flat bottomed plates pre-coated with 60 μL/well Geltrex. Cells were immediately placed within the humidified LivecyteCell Imaging chamber (Phasefocus) maintained at 37°C 5% CO 2 in air and left to equilibrate for 20 min. (A) A single 1 mm 2 region per well was imaged with a 10× objective every hour for 10 h. Integrated angiogenesis network analysis was performed using PhaseFocus Cell Analysis Toolbox version 3.8.1 on each well over the total length of the assay. Images shown are for the 10 h time point. (B) Example of the analysis with individual cells indicated by multicolored shapes and tube networks shown in yellow. (C) The total length of the network (μm) was calculated on a per cell basis at the 10 h time point. Data are expressed as the mean ± S.E.M of 4 independent experiments (4–5 wells analyzed per cell line, per experiment).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: CRISPR, Expressing, Imaging

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Effect of histamine and VEGF 165 a on HiBiT E-selectin expression in gene-edited TERT2-HUVEC clone C8 (A and B) Increase in luminescence under basal conditions and in response to 8 h stimulation with (A) 100 nM histamine or (B) a combination of 100 nM histamine and 1 nM TNFα in HiBiT genome-edited clone C8 TERT2-HUVECs. (C and D) Increase in luminescence under basal conditions and in response to 8 h stimulation with 100 nM VEGF 165 a (C) or (D) a combination of 100 nM VEGF 165 a and 1 nM TNFα in HiBiT genome-edited clone C8 TERT2-HUVECs. Values represent mean ± S.E.M of the five independent experiments, each performed in triplicate. ∗p < 0.05 compared to vehicle control (paired t test). ∗∗p < 0.01 compared to vehicle control or #p < 0.05 compared to TNFα alone (One-way ANOVA).

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet: Kinetic profile of TNFα-stimulated luminescence in clonal HiBiT-edited E-selectin TERT2-HUVECs To monitor the real-time kinetics of TNFα-induced expression of E-selectin, experiments were undertaken with the homozygous TERT2-HUVEC HiBiT E-selectin clone C8. As these kinetic experiments were performed over 15 h, the long acting caged nanoluciferase substrate endurazine was used. (A) Time course of HiBiT E-selectin expression over 15 h in the continued presence of 50 nM LgBiT and endurazine. Values represent mean ± S.E.M from five independent experiments. (B) Incubation with 1 nM TNFα in the continued presence of 50 nM LgBiT and endurazine yielded a significant increase in luminescence over 8 h (∗∗∗p < 0.001; paired t test of 5 independent experiments). (C) To investigate potential depletion of LgBiT through the experiment, experiments were either performed in the continuous presence of 50 nM LgBiT incubation or following a 20 min addition of LgBiT (50 nM) at the end of the 15 h incubation with 1 nM TNFα. There was no significant difference between the luminescence values obtained. (D) Time course of HiBiT E-selectin expression in response to increasing concentrations of TNFα. Values represent mean ± S.E.M from six (or five, 0.25 nM) independent experiments. (E) Peak responses from (D) expressed as a percentage of the response to 1 nM TNFα. (F) Log peak responses to increasing concentrations of TNFα. Data taken from (E). ∗∗∗∗p < 0.0001 (one-way ANOVA with Dunnett multiple comparison test). Symbols show the individual means in 5 or 6 independent experiments.

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Expressing, Incubation

Journal: iScience

Article Title: Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

doi: 10.1016/j.isci.2023.107232

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were mouse monoclonal anti-HiBiT (N7200, Promega; 1:1000 dilution), mouse monoclonal anti-E-selectin (CD62E; 1:1000 dilution) and rabbit anti-α-tubulin (2114; Cell Signalling; 1:1000 dilution).

Techniques: Produced, Recombinant, Transfection, Modification, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Blocking Assay, Staining, Purification, Electroporation, Luciferase, Plasmid Preparation, Software