Journal: Frontiers in Oncology

Article Title: PALB2 deficiency may sensitize H3K27M-mutant pediatric HGG cells to BMN673/talazoparib

doi: 10.3389/fonc.2025.1589396

Figure Lengend Snippet: Analysis of NHEJ activity via the EJ5 reporter assay in SF188 and Res259 cells stably expressing the H3 WT or H3K27M mutation. (A) Schematic representation of the EJ5 reporter for NHEJ repair. EJ5-GFP is shown along with two classes of NHEJ repair products that can restore a GFP expression cassette. In the starting construct, the GFP gene is inactive. GFP expression is only activated when I-Sce1-induced DSBs are successfully repaired via NHEJ. (B) H3K27M-mutant SF188 cells and WT SF188 cells containing the reporter cassette were transfected with the I-SceI and DsRed plasmids. Left panel: Immunofluorescence images of GFP-positive cells and DsRed-positive cells (scale bars, 100 µm). Right panel: NHEJ activity in H3K27M-mutant SF188 cells and WT SF188 cells. (C) H3K27M-mutant Res259 cells and WT Res259 cells containing reporter cassettes were transfected with I-SceI and DsRed plasmids. Left panel: Immunofluorescence images of GFP-positive cells and DsRed-positive cells (scale bars, 100 µm). Right panel: NHEJ activity in H3K27M-mutant Res259 cells and WT Res259 cells. NHEJ activity was determined by normalizing the percentage of eGFP-positive cells to the percentage of DsRed-positive cells. The data are shown as the mean ± SD from three independent experiments. Student’s t test was used to calculate p values. ** p < 0.01 versus the corresponding control.

Article Snippet: The indicated EJ5 reporter cell lines were transfected with two plasmids, namely, the I-SceI expression vector pCBASce (Addgene #26477) and the DsRed expression vector (Addgene #54493), 48 h later, the percentage of GFP-positive cells among DsRed-positive cells was evaluated by imaging with a Carl Zeiss LSM 710 confocal fluorescence microscope and quantified via FACS on a BD AccuriTM C6 flow cytometer (BD Biosciences, USA) using BD AccuriTM software.

Techniques: Activity Assay, Reporter Assay, Stable Transfection, Expressing, Mutagenesis, Construct, Transfection, Immunofluorescence, Control