i scei expression plasmid  (Addgene inc)

Journal: Molecules and Cells

Article Title: Tumor-derived CD84 promotes growth of acute myeloid leukemia cells via regulating nonhomologous DNA end-joining pathway

doi: 10.1016/j.mocell.2025.100253

Figure Lengend Snippet: Knockdown of CD84 expression inhibits NHEJ repair in AML cells. (A) Characterization of the influence of CD84 on NHEJ repair by pimEJ5-EGFP. Design of the NHEJ assay. Transiently expressed I-SceI protein cleaves the I-SceI sites and produces DSBs with incompatible ends in the substrate. NHEJ repair of 2 broken DNA ends results in EGFP expression. (B) Shown are representative flow cytometry plots. (C) The percentage of EGFP+ cells was summarized. n = 4, mean ± SD, multiple t test, *** P < .0001 compared with scramble. (D-F) The DSBs damage accumulation was analyzed in scramble and shRNA-infected cells by confocal immunofluorescence analysis of γ-H2AX foci at indicated time post irradiation (IR). Shown are representative images in THP-1 cells (D) (Red, γ-H2AX; Blue, DAPI; Bar, 5 µm). The average numbers of γ-H2AX foci per cell at indicated time post IR were counted (E) and (F). n = 50 cells per group, mean ± SD, 2-way ANOVA test, *** P < .0001. (G-H) Expression of BCL-2 and cleaved CASPASE3 (c-CAS3) was detected in CD84 shRNA-infected cells (H) or B4 antibody-treated cells (I). (I) Quantification of the log2 (fold change) expression of p-AKT, BCL-2, and c-CAS3 in (H) and (I). All experiments were repeated in 3 independent biological replicates.

Article Snippet: To induce DSBs, the I-SceI expression plasmid pCBA SceI (#26477, Addgene) along with either scramble or CD84 shRNA plasmid was transfected into 293T cells overexpressing pEJ5-GFP.

Techniques: Knockdown, Expressing, NHEJ Assay, Flow Cytometry, shRNA, Infection, Immunofluorescence, Irradiation