Journal: International Journal of Molecular Sciences

Article Title: Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

doi: 10.3390/ijms252011320

Figure Lengend Snippet: Validation analysis of HeLa cell clones with integrated rhBCHE. ( A ) Schematic diagram of the rhBCHE template. ( B ) Circularization system of the rhBCHE template. Yellow and blue arrows represent multimolecular and monomolecular products, respectively, in the 5 ng/μL circularization system. ( C ) Western blot analysis. After ultrafiltration, the cell supernatant was analyzed by Western blot using different amounts of rhBChE standards as control. ( D ) Deglycosylation assay of the supernatant protein. The molecular weight of the supernatant protein decreased following deglycosylation with rhBChE standard. ( E ) qPCR detection of BCHE transcription levels. ( F ) Analysis of supernatant protein activity. The activity of supernatants protein after ultrafiltration from different cells was determined using the Ellman assay. ( G ) Determination of anti-DDVP activity. A 50 μm concentration was determined as the median lethal concentration dose for WT HeLa cells, and cell viability in other clones was measured relative to this (N = 4). ( C , E , G ) Data are presented as the mean ± SD from three or four technical replicates. Statistical analysis was performed using an unpaired Student’s t -test. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.

Article Snippet: Recombinant human BChE (Sinobiological, Beijing, China) was used as a control.

Techniques: Clone Assay, Western Blot, Control, Molecular Weight, Activity Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

doi: 10.3390/ijms252011320

Figure Lengend Snippet: Detection of HEK-293T cell clones with double-copy integration of rhBCHE. ( A ) PCR genotyping of the rhBCHE-forward clones targeting the AAVS1 and GRIK1 loci in HEK-293T cells. The lanes within red rectangles indicate forward knock-in patterns, while the red asterisks indicate reverse knock-in patterns at the GRIK1 locus. ( B ) Western blot analysis. AF44: clone with single-copy forward integration of rhBCHE at the AAVS1 locus; AF44-GF5 and AF44-GF10: clone with single-copy forward integration of rhBCHE at both the AAVS1 and GRIK1 loci; AF44-GR6: clone with single-copy forward integration of rhBCHE at the AAVS1 locus and single-copy reverse integration of rhBCHE at the GRIK1 locus. Tubulin served as the loading control. ( C ) Deglycosylation of the intracellular protein was analyzed by Western blot with GAPDH as the loading control. ( D ) Deglycosylation of the supernatant protein was analyzed by Western blot. ( E ) Multimerization of the supernatant protein was detected by Western blot. The blue arrow indicates the dimer protein band, and the green arrow indicates the monomer protein band. ( F ) Ellman activity assay of the supernatant protein. The rhBChE activity of AF44-GR6 was significantly higher than that of AF44. Data are presented as the mean ± SD from three technical replicates. An unpaired Student’s t -test was used for statistical analysis. (*) p < 0.05.

Article Snippet: Recombinant human BChE (Sinobiological, Beijing, China) was used as a control.

Techniques: Clone Assay, Knock-In, Western Blot, Control, Activity Assay

Journal: Annals of Medicine

Article Title: Organoids from patient biopsy samples can predict the response of BC patients to neoadjuvant chemotherapy

doi: 10.1080/07853890.2022.2122550

Figure Lengend Snippet: The main components of human PDO culture medium.

Article Snippet: Noggin , Peprotech , 120-10C-50 , 100 ng·ul −1 , PBS , 100 ng·ml −1.

Techniques: Concentration Assay, Recombinant