nhej reporter plasmid pimej5 gfp  (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: CD84 regulates multiple signal transduction pathways in AML cells. (A) CD84 co-expression genes were analyzed in 3 AML independent datasets in cBioPortal, by using P value < .05 as the cutoff criteria. CD84-negative co-expression genes (934 genes, upper panel) and CD84-positive coexpression genes (1549 genes, lower panel) were analyzed by Venn diagrams. (B) The 1549 positive coexpression genes of CD84 were subjected to GO enrichment analysis. The most enriched biological process (BP), molecular function (MF), and cellular component (CC) categories are plotted. (C) The top 10 KEGG pathways with the highest enrichment of CD84-positive coexpression genes. (D) Summary of the Spearman's correlation coefficient between CD84 and the genes that enriched in nonhomologous end-joining in panel C.

Article Snippet: Briefly, the NHEJ reporter plasmid pimEJ5-GFP (#44026, Addgene), containing a promoter separated from an EGFP coding cassette by a puro gene flanked by 2 I-SceI sites, was transfected into 293T cells using polyJet.

Techniques: Transduction, Expressing

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: CD84 regulates expression of NHEJ core factors. (A-D), Shown are the Spearman correlation scatterplots between CD84 and LIG4 (E), PRKDC (F) , XRCC5 (G), and DCLRE1C (H) . n = 173, Spearman’s rank test. (E) Quantitative RT-PCR detected the mRNA expression of the indicated genes in scramble or CD84 shRNA-infected MV4-11 cells. n = 3, mean ± SD, multiple t test, *** P < .0001. F, AML cells were treated with CD84-stimulating antibody, followed by Western blotting and CO-IP. G-H, Expression of AKT and p-AKT was detected in CD84 shRNA-infected cells (G) or B4 antibody-treated cells (H). (I-J) Cells treated with CD84-stimulating antibody or AKT inhibitor, followed by Western blotting (I) and RT-PCR (J).

Article Snippet: Briefly, the NHEJ reporter plasmid pimEJ5-GFP (#44026, Addgene), containing a promoter separated from an EGFP coding cassette by a puro gene flanked by 2 I-SceI sites, was transfected into 293T cells using polyJet.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Infection, Western Blot, Co-Immunoprecipitation Assay, Reverse Transcription Polymerase Chain Reaction

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: Briefly, the NHEJ reporter plasmid pimEJ5-GFP (#44026, Addgene), containing a promoter separated from an EGFP coding cassette by a puro gene flanked by 2 I-SceI sites, was transfected into 293T cells using polyJet.

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

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: CD84 supports growth of human acute myeloid leukemia-initiating cells. (A) Relative expression of CD84 in different hematopoietic or myeloid compartments ( GSE30029 ). Normal CD34+ n = 22, AML CD34− n = 37, AML CD34+ n = 38, mean ± SD, multiple t test, * P < .05, *** P < .0001. (B) Representative flow cytometric analysis of CD84 expression on the immunophenotypic CD34+CD38− LICs and CD34−CD38− differentiated AML cells. (C) Quantification of the mean fluorescence intensities for CD84 expression on the indicated leukemia cells in panel B. n = 5 patients, mean ± SD, paired Student’s t test, ** P < .001. (D) Representative images of the colonies formed by the CD84 high and CD84 low PBMC cells after 10 days of culture. Bar, 50 µm. (E) and (F) Quantification of colony numbers (E) and cell numbers (F) in (D). n = 3, mean ± SD, Student’s t test, * P < .01; *** P < .0001. (G) Representative images of the colonies formed by the scramble and CD84 shRNA-infected PBMC cells after 10 days of culture. Bar, 50 µm. (H) and (I) Quantification of colony numbers (H) and cell numbers (I) in panel G. n = 3, mean ± SD, multiple t test, ** P < .001; *** P < .0001. (D-I), Experiments were performed using samples from 2 different AML patients, and results from 1 representative patient are shown here. (J) Proposed working model of how CD84 regulates LICs and leukemic cells. Suppression of CD84 leads to downregulation of NHEJ core factors (eg, PRKDC , LIG4 , and XRCC5 ), thereby impairing NHEJ repair efficiency. The functional impairment of NHEJ results in the accumulation of DSBs, which subsequently triggers cell apoptosis and inhibits the self-renewal of LICs.

Article Snippet: Briefly, the NHEJ reporter plasmid pimEJ5-GFP (#44026, Addgene), containing a promoter separated from an EGFP coding cassette by a puro gene flanked by 2 I-SceI sites, was transfected into 293T cells using polyJet.

Techniques: Expressing, Fluorescence, shRNA, Infection, Functional Assay