Figure 4 from Integrated Genetic Screening Identifies FEN1 as a Selective Vulnerability and Temozolomide Resistance Mediator in Glioblastoma Stem Cells
FEN1 inhibition enhances TMZ-induced DNA damage. A, Immunoblotting of FEN1i-resistant and FEN1i-sensitive GSCs for FEN1 protein after treatment with 100 μmol/L TMZ (n = 3 biological replicates per group). B, Immunoblotting of patient-matched primary (FEN1i-resistant; BT594) and recurrent (FEN1i-sensitive; BT972) GSCs for FEN1 protein after treatment with DMSO (control) or 100 μmol/L TMZ. FEN1 signal intensity normalized to vinculin control is shown for the representative immunoblot. C, Scatterplots indicating representative individual cellular comet tail moment values of FEN1i-resistant (BT594) and FEN1i-sensitive (BT972) GSCs from each corresponding cell/treatment type: DMSO alone (control), TMZ (200 μmol/L), FEN1i (30 μmol/L), or combination (TMZ–FEN1i, 200 μmol/L/30 μmol/L). D, Quantification of comet assays. Relative tail movement of FEN1i-resistant and FEN1i-sensitive GSCs is shown with increasing concentrations of TMZ ± 30 μmol/L FEN1 inhibitor for 48 hours. Data are represented as mean ± SE; n = 12 experimental replicates. E, Immunoblotting of FEN1i-resistant (BT594) and FEN1i-sensitive (BT972) GSCs for phosphorylated CHK1, total CHK1, phosphorylated CHK2, and total CHK2 after a 3-day treatment with DMSO (control), TMZ (45 μmol/L), FEN1i (30 μmol/L), or combination (TMZ and FEN1i). F, Quantification of immunoblotting in E. The proportion of phosphorylated (active) CHK1 and CHK2 is shown, with signal intensities normalized to vinculin control. Quantification is provided from the representative immunoblot shown to facilitate comparison across the indicated treatment conditions; independent replicate immunoblot experiments were not performed for this analysis. G, Immunoblotting of FEN1i-resistant and FEN1i-sensitive GSCs (n = 3 biological replicates per grouping) for total CHK1 and total CHK2 protein after treatment with TMZ (100 μmol/L). Heatmap color corresponds to normalized FEN1 intensity. All samples of each individual experiment were run on the same immunoblot under identical experimental conditions, with immunoblots cropped for presentation. Quantification of immunoblots is presented for visualization of relative signal intensity only and should not be interpreted as statistical comparisons across experiments. ns, not statistically significant. **, P < 0.01; ***, P < 0.001.