Journal: Frontiers in Microbiology

Article Title: The gene YEF3 function encoding translation elongation factor eEF3 is partially conserved across fungi

doi: 10.3389/fmicb.2024.1438900

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: Recoded eEF3-HA DNAs (GeneScript) were subcloned from pUC57-simple onto the p301- TRP1 vector.

Techniques: Plasmid Preparation, Cloning, PCR Cloning, Marker, Expressing, Clone Assay, Sequencing

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Schematic design of F3-Gel and proof of production. (A) Schematic design of pET-F3-Gel vector. The pET-F3-Gel vector was constructed by inserting the N-terminal sequence of gelonin and F3-gelonin gene into a pET-TRX vector containing the thioredoxin (TRX) gene. (B) Schematic images of TRX-F3-Gel, F3-Gel and Gelonin. (C) SDS-PAGE results of Ni-NTA and heparin column purification of TRX-F3-Gel. Lane M: markers of the protein molecular weight standard (Mark 12TM standard, Invitrogen). Lane NTA-Ni: TRX-F3-Gel purified by a Ni-NTA column. Lane Hep: fraction obtained by heparin column purification of F3-Gel after cleavage of thioredoxin-6×His tag from the TRX-F3-Gel. Lane WB: Western blot assay results of TRX-F3-Gel. The purity of the final F3-Gel product was determined based on densitometry analysis using ImageJ software. TRX-F3-Gel: recombinant thioredoxin-6×His tagged-F3-gelonin fusion protein; F3-Gel: recombinant F3-gelonin fusion protein, Gelonin: recombinant gelonin.

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Plasmid Preparation, Construct, Sequencing, SDS Page, Purification, Molecular Weight, Western Blot, Software, Recombinant

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Protein synthesis inhibition by F3-Gel. Inhibition of protein translation by unmodified gelonin (Gelonin) and recombinant F3-gelonin fusion protein (F3-Gel) was determined using a cell-free translational system and luciferase as the marker. The quantity of the translated luciferase was measured using a chemiluminescent assay (mean±SEM; n=3). Luminescence intensity vs gelonin concentration curves were fitted by a nonlinear regression model using Prism software.

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Inhibition, Recombinant, Luciferase, Marker, Concentration Assay, Software

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Cell uptake study results of gelonin and F3-Gel on 293 HEK noncancerous cells and U87 MG cancer cells. (A) Confocal microscopy images of live cells after incubation with either TRITC-labeled gelonin or F3-Gel for 4 h at 37 °C. After incubation, the cells were washed three times with PBS, the nuclei were counterstained with Hoechst 33342, and after being washed with PBS, the cell images were acquired with a confocal microscope utilizing Hoechst (H; blue) and rhodamine (R; red) channels. Scale bar indicates 40 μm. (B) Quantitative analysis of cellular uptake of gelonin or F3-Gel in U87 MG and 293 HEK cells (mean±SEM. n=3. *P<0.05, **P<0.01 by t-tests). Fluorescence intensity values per 106 cells are depicted over the incubation time period (until 4 h).

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Confocal Microscopy, Incubation, Labeling, Microscopy, Fluorescence

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Fluorescence intensity values (×10 4 ) related with the intracellular level of gelonin and F3-Gel into various cancer and noncancerous cell lines.

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Fluorescence

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Cytotoxicity assay results. The viability profiles of cancer (HeLa, LnCaP, 9L and U87 MG) and noncancerous (293 HEK and SVGp12) cells were evaluated after 48 h of incubation with varied concentrations (10−11–10−4 μmol/L) of either gelonin or F3-Gel. Plots are depicted with the mean±SEM (n=3).

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Cytotoxicity Assay, Incubation

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Cytotoxicity levels (IC 50 ) of gelonin and F3-Gel.

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques:

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Intracellular activity of F3-Gel in U87 MG cells. Relative cellular protein levels in U87 MG cancer cells were measured after 24 h of incubation with gelonin or F3-Gel (at 0.1, 0.5 or 1 μmol/L). Control cells were incubated with PBS buffer. Plots are depicted with the mean±SEM (n=3).

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Activity Assay, Incubation, Control

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Organ distribution profiles of gelonin and F3-Gel in U87 MG sc xenograft tumor-bearing mice. (A) Biodistribution imaging of organs and (B) weight-normalized mean fluorescence intensity (MFI) values measured from each organ after administration with DylightTM775-B4-labeled gelonin. (C) Bioimaging results of organs and (D) weight-normalized MFI values after the administration of labeled F3-Gel via the tail vein. At the indicated time points (1, 4, and 16 h), major organs were harvested and imaged using an IVIS® spectrum imaging system equipped with an ICG filter (Ex/Em: 745/800 nm). Fluorescence intensities were observed in each organ up to 16 h, and MFI values were normalized by organ weights. Plots are depicted with the mean±SEM (n=6).

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Imaging, Fluorescence, Labeling

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: Tumor distribution profiles from the U87 MG xenograft mice until 16 h after administration with gelonin or F3-Gel. Weight-normalized mean fluorescence intensities in the tumor are displayed for both gelonin samples with the mean±SEM (n=6).

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: Fluorescence

Journal: Acta Pharmacologica Sinica

Article Title: Molecular tumor targeting of gelonin by fusion with F3 peptide

doi: 10.1038/aps.2017.20

Figure Lengend Snippet: In vivo evaluation study of the therapeutic efficacy and toxicity of gelonin or F3-Gel in U87 MG xenograft tumor-bearing mice. U87 MG sc xenograft tumor-bearing mice were randomly divided into six groups when the tumor size reached an average of 200 mm3 (d 11 after administration). (A) Tumor volumes (mm3) and (B) body weight change (%) in mice at d 11, 14, 17, 20, 23, and 25 after tail vein injection of gelonin samples with either 1) PBS, 2) gelonin (0.75 μmol/kg), 3) F3-Gel (0.125 μmol/kg), 4) F3-Gel (0.25 μmol/kg), 5) F3-Gel (0.5 μmol/kg) or 6) F3-Gel (0.75 μmol/kg). The mice were treated a total of three times with the agents (1–6) at d 11, 14 and 17 via tail vein injection. The tumor volume and body weight of the mice were measured until the average tumor size of the PBS control group reached 1000 mm3. Mean±SEM (n=6). One-way ANOVA with Tukey's multiple comparison test as the post hoc test; *P<0.05, **P<0.01.

Article Snippet: Genes encoding a portion of the N-terminal region of gelonin and an F3 peptide (299 bp; GenScript USA Inc, Piscataway, NJ, USA) were double digested ( Nde I and Nhe I) from a pUC-57 simple vector, and the gene fragments were separated on a 1% agarose gel, purified, and ligated into a linearized pET22b-TRX-Gel vector previously prepared in our lab 24 .

Techniques: In Vivo, Drug discovery, Injection, Control, Comparison

Journal: Cell & Bioscience

Article Title: Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells

doi: 10.1186/s13578-018-0200-z

Figure Lengend Snippet: The enhancement of mutation-correction efficiency of a GFP-silent mutation by ssODN and SCR7 treatment in MCF-7/GFP-Mut cells. MCF-7/GFP-Mut cells were co-transfected with the pCS2-Cas9-U6-sgRNA (GFP-Mut) vector or/and GFP ssODN. After transfection, cells were treated with vehicle control or SCR7 (20 μM) for 48 h. At the end of experiment, the GFP-positive cells were quantified by FACS. MCF-7 cells transfected with the wild-type GFP vector (pSIN-EF1-GFP-puromycin) were used as GFP-positive control cells. a – g show the representative flow cytometric analyses of MCF-7 cells transfected with the wild-type GFP vector ( a ), MCF-7/GFP-Mut cells without transfection ( b ), MCF-7/GFP-Mut cells transfected with pCS2-Cas9-U6-sgRNA ( c ), or GFP ssODN alone ( e ), MCF-7/GFP-Mut cells treated with 20 μM SCR7 alone ( d ), and MCF-7/GFP-Mut cells transfected with both pCS2-Cas9-U6-sgRNA and GFP ssODN without ( f ), or with 20 μM SCR7 treatment ( g ). h shows a quantitation of GFP-positive cells in percentage (mean ± SD) from 3 independent experiments. SSC—side scatter. *p < 0.05, **p < 0.01

Article Snippet: Briefly, to create a sgRNA expression vector, we placed a U6 promoter followed by two BbsI sites upstream of the recently described sgRNA scaffold [ ], which was synthesized by GenScript and cloned into the pUC57-Simple vector [ ].

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Control, Positive Control, Quantitation Assay