pcmv3 survivin expression plasmid  (Sino Biological)

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: Communications Biology

Article Title: ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways

doi: 10.1038/s42003-026-09604-z

Figure Lengend Snippet: a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and pReporter–Donor. DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.

Article Snippet: To prepare dsDNA, the pReporter–donor plasmid was digested with HindIII and AflII (NEB).

Techniques: Residue, Fluorescence, Construct, Sequencing, Plasmid Preparation, Transfection, Expressing, Control, Knock-In, CRISPR