human embryonic kidney derived hek293t cells  (Procell Inc)

Journal: Vaccines

Article Title: A Pool of Ferritin Nanoparticles Delivering Six Proteins of African Swine Fever Virus Induces Robust Humoral and Cellular Immune Responses in Pigs

doi: 10.3390/vaccines14010093

Figure Lengend Snippet: Evaluation of the immunogenicity of the antigen candidates in pigs. ( A ) Schematic diagram of pig vaccination. Each pig was vaccinated with 100 μg of antigens at days 0, 14, and 28. Blood was collected at 0, 14, 28, and 42 days post-vaccination (dpv). ( B ) Verification of eukaryotic expression plasmids by Indirect immunofluorescence assay (IFA). HEK293T cells were transfected with plasmid pCAGGS-p30, pCAGGS-p54, pCAGGS-pE120R, pCAGGS-pH124R, pCAGGS-pE184L or pCAGGS-CD2v. At 24 h post-transfection, the cells were lysed with RIPA buffer and the whole cell lysates were verified by Western blotting with an anti-Flag monoclonal antibody. ( C ) IFA for ASFV antigen-specific antibody detections. By detecting the sera at 42 dpv to determine whether the antibody turned positive. Sera collected at 0 dpv were the negative control, and anti-Flag antibody was used as the positive control. ( D ) Antigen-specific antibody titers were determined by in-house indirect enzyme-linked immunosorbent assay (iELISA) for p30, p54, pE120R, pH124R, pE184L, and CD2v at 0 and 42 dpv. The dashed lines represent the cut-off value for each iELISA. Group 1: pigs vaccinated with p30 (1-1, 1-2 and 1-3; n = 3); Group 2: pigs vaccinated with p54 (2-1, 2-2 and 2-3; n = 3); Group 3: pigs vaccinated with pE120R (3-1, 3-2 and 3-3; n = 3); Group 4: pigs vaccinated with pH124R (4-1 and 4-2; n = 2); Group 5: pigs vaccinated with pE184L (5-1 and 5-2; n = 2); Group 6: pigs vaccinated with CD2v (6-3; n = 1). The reduced sample sizes in Groups 4-6 were due to unintended infection, and data from these groups should be interpreted as indicating immunological trends rather than definitive outcomes.

Article Snippet: Human embryonic kidney-derived HEK293T cells (Procell, Wuhan, China) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS.

Techniques: Immunopeptidomics, Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Western Blot, Negative Control, Positive Control, Indirect ELISA, Infection

Journal: STAR Protocols

Article Title: A micropattern-based assay to study contact inhibition of locomotion and entosis of adherent human and canine cells in vitro

doi: 10.1016/j.xpro.2023.102186

Figure Lengend Snippet: Figure 1

Article Snippet: HEK293T: human epithelial-like cells derived from a female embryonic kidney , ATCC , #CRL-3216 RRID: CVCL_0063.

Techniques: Virus, Recombinant, Transfection, Polymer, Derivative Assay, shRNA, Plasmid Preparation, Software, Microscopy

Journal: STAR Protocols

Article Title: A micropattern-based assay to study contact inhibition of locomotion and entosis of adherent human and canine cells in vitro

doi: 10.1016/j.xpro.2023.102186

Figure Lengend Snippet: HEK293T cell medium

Article Snippet: HEK293T: human epithelial-like cells derived from a female embryonic kidney , ATCC , #CRL-3216 RRID: CVCL_0063.

Techniques: Concentration Assay

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: Over-expression of STYK1/NOK causes mitotic abnormality. (A) The endogenous STYK1/NOK expressions in different cell lines by immunoblot analysis in PC-3 cells, U-87 MG cells, DLD-1 cells, HeLa cells, HEK293T cells, RPE-1 cells and HCT116 cells. GAPDH was used as a loading control. The band intensities of STYK1/NOK were quantified with FIJI software and then normalized to that of GAPDH. The relative expressions of all others were compared to that of DLD-1 cells of which was set as 1.00. The original blots are presented in Supplementary Figure 1A and 1B. (B) Establishment of the doxycycline-controlled STYK1/NOK stable cell line. HeLa cells were infected with lentiviral constructs containing doxycycline-inducible STYK1/NOK expression cassettes. The infected cells were selected with 1 μg/mL of puromycin for 5 days to obtain the stable cell line Tet-S/N. Tet-S/N cells were treated with doxycycline for 48 h before harvesting. The original blots are presented in Supplementary Figure 1C. (C) Representative immunofluorescent images of the established stable HeLa cells that were stained with α-tubulin (Green) and DAPI (Blue). Arrows indicated cells undergoing cytokinesis. Quantification of immunofluorescent results presented in (C) by calculating proportions of mitotic cells (D) and cells undergoing cytokinesis (E) to the total cells counted. The data were mean ± SEM from four independent experiments. (F) Immunoblot analysis on doxycycline-induced STYK1 silencing. DLD-1 cells were stably transfected with lentiviral doxycycline-controlled lentiviral constructs expressing shSTYK1/NOK to establish the Tet-shS/N cell line. The transfected cells were selected with 2 μg/mL of puromycin for 5 days. Cells were cultured in the presence and absence of doxycycline for 3 days before harvesting. The original blots are presented in Supplementary Figure 1D. (G) Representative immunofluorescent images of the established stable Tet-shS/N cells that were stained with α-tubulin (Green) and DAPI (Blue). Quantification of immunofluorescent results showed the proportions of mitotic cells (H) and cytokinesis (I) as mean ± SEM from three independent experiments. Mitotic cells were distinguished and counted by morphological evaluation based on the α-tubulin and DAPI staining. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Over Expression, Western Blot, Control, Software, Stable Transfection, Infection, Construct, Expressing, Staining, Transfection, Cell Culture

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: CDH1 interacts with STYK1/NOK in a kinase domain dependent manner. (A) HeLa cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 or an empty vector. Cells were treated with 50 μg/mL of cycloheximide for the indicated time before harvesting. Immunoblot analysis (upper panel) was conducted to detect the expression of STYK1/NOK. The expression of GAPDH served as an internal control. The original blots are presented in Supplementary Figure 4A. Quantification analysis (lower panel) of the relative band intensity of STYK1/NOK was performed to determine the protein half-life of STYK1/NOK. The STYK1/NOK band intensities were normalized to that of GAPDH, then further normalized to that of t = 0 time point. (B) HEK293T cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 and treated with 10 μM of MG132 for 4 h before harvesting. Co-immunoprecipitation for detection of STYK1/NOK and CDH1 interaction was then performed. The original blots are presented in Supplementary Figure 4B. (C) Schematic representation of the wild type STYK1/NOK domain structure and its five D-boxes (D1 to D5). Red letters represent conserved residues and green letters represent preferred residues. (D) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its D-box deleted mutants (ΔD1 to ΔD5) and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation was carried out to detect the interaction between CDH1 and either STYK1/NOK or its mutants. The original blots are presented in Supplementary Figure 4C. (E) Schematic presentation of the full-length STYK1/NOK and its truncated forms. (F) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its truncated forms and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation assay was used to detect the interaction between CDH1 and full-length STYK1/NOK or its truncated forms. The star ∗ represents non-specific proteins. The original blots are presented in Supplementary Figure 4D.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: Loss of contact inhibition of locomotion in the absence of JAM-A promotes entotic cell engulfment

doi: 10.1016/j.isci.2022.105144

Figure Lengend Snippet:

Article Snippet: HEK293T: human epithelial-like cells derived from a femal embryonic kidney , ATCC , #CRL-3216 RRID: CVCL_0063.

Techniques: Virus, Recombinant, Isolation, Clinical Proteomics, Imaging, Derivative Assay, shRNA, Software

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Commercial cell lines Human female embryonic kidney derived HEK293T and Mouse male Neuro-2a acquired from ATCC were used for MADR in vitro validation in human and mouse cells respectively.

Techniques: Clone Assay, Recombinant, Lysis, Sequencing, Plasmid Preparation, Mutagenesis, Software

Journal: Nucleic Acid Therapeutics

Article Title: Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells

doi: 10.1089/nat.2018.0743

Figure Lengend Snippet: Transient transfection of amiRNAs and their effect on cell viability. (A) Cells seeded in a 96-well plate were infected with JEV at a MOI 5. Three hours postinfection, the cells were transfected with three different concentrations of amiRNAs (100, 500, and 1,000 ng) of single amiRNA per well. After 48 hpi, MTT reagent was added, and absorbance was measured at 570 nm. Results represent three independent experiments. (B) Cells were seeded in a 6-well plate and were transfected with four different concentrations of amiRNAs (50, 250, 500, and 1,000 ng) of single amiRNA per well. After 24 h, amiRNAs expression was monitored by checking eGFP expression under a fluorescence microscope. Representative images of amiRNA-treated HEK293T cells at 10 × magnification are shown. (C) RT-PCR analysis of four ISG ( Ifitm1 , Oasl1 , Oasl2 , and Isg15 ) mRNA expression. Gapdh gene expression was used to normalize the ISG expression. emGFP, emerald green fluorescent protein; hpi, hours postinfection; IFN, interferon; ISGs, IFN-stimulated genes; MOI, multiplicity of infection; qRT-PCR, quantitative real time polymerase chain reaction. Color images available online at www.liebertpub.com/nat

Article Snippet: Porcine stable (PS) kidney cell line, mouse neuroblastoma cells (N2a), and human embryonic kidney-derived cell line (HEK293T) were procured from National Centre for Cell Science, Pune, India.

Techniques: Transfection, Infection, Expressing, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Nucleic Acid Therapeutics

Article Title: Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells

doi: 10.1089/nat.2018.0743

Figure Lengend Snippet: Binding of amiRNAs on 3 ′ UTR of JEV Vellore strain (P20778) HEK293T cells were cotransfected with 100 ng JEV3 ′ UTR luciferase construct and different concentration of amiRNA-expressing vector. After 48 h post-transfection, cells were lysed, and luciferase activity was measured. The data are shown as the firefly luciferase activity relative to the Renilla luciferase activity and are representative of three independent experiments. (A) Results represent the binding effect of 1,000 ng of amiRNA construct on JEV 3 ′ UTR luciferase construct. The asterisk indicates statistical significance at 48 hpi (* P < 0.05). (B) Results represent the binding effect of different doses of amiRNA construct (50, 250, 500, and 1,000 ng) on JEV-3 ′ UTR luciferase construct. (C) The nucleotide sequence of amiRNA #1, amiRNA #2, and amiRNA #5 ( left panel ) and their target site in JEV 3 ′ UTR ( right panel ).

Article Snippet: Porcine stable (PS) kidney cell line, mouse neuroblastoma cells (N2a), and human embryonic kidney-derived cell line (HEK293T) were procured from National Centre for Cell Science, Pune, India.

Techniques: Binding Assay, Luciferase, Construct, Concentration Assay, Expressing, Plasmid Preparation, Transfection, Activity Assay, Sequencing