Fig 1: IRP1 knockdown reduces IR-induced protein peroxide content.Immediately following IR exposure at indicated doses, cells were washed and then frozen in −80°C ethanol before processing lysates for protein peroxide content by the PCA-FOX assay. Data are plotted as pmol of peroxide per microgram of protein from a hydrogen peroxide standard curve. Data are mean +/− SEM of a representative experiments performed three times. Student's t-test was used to test significance (* = p<0.05).
Fig 2: IR-induced apoptosis is reduced and clonogenic survival following low LET IR is improved in IRP1- cells.Cells were exposed to indicated doses of gamma IR (panel a, c, & d) or alpha IR (panel b) and assayed for apoptosis by annexin V & PI staining (panels a–b) at 48 h post-IR, Alamar Blue assay at 48 h post-IR (panel c), or executioner caspase activity at 24 h post-IR (panel d). Data are mean +/− SEM of at least two independent experiments. Two-way repeated measures ANOVA was used to measure statistical significance at each dose (** = p<0.01, *** = p<0.001). Panels e–f: clonogenic survival assays approximately 14 d following gamma IR (panel e) or alpha IR (panel f). Data are mean +/− SEM of three independent experiments. Curve fits compared by a paired t-test were found to be significantly different following gamma IR (p<0.01) but not alpha IR.
Fig 3: Repair, but not initiation, of DSBs is more rapid in IRP1- cells.Panel a: the increase in the number of foci-positive cells (those containing greater than 10 per nucleus) versus control and 2 Gy irradiated cells were scored at 30 m and 4 h post-IR and are plotted as percent foci-positive at 4 h post-IR versus 30 m. Data are mean +/− SEM from two independent experiments stained simultaneously. Exactly 500 cells were scored per sample. The paired t-test p-value was 0.07. Panel b: cells were irradiated with 0 or 4 Gy gamma rays and immediately assayed for DSB formation by the neutral comet assay. Whiskers represent the 1st and 99th percentiles, and means are indicated by ‘+’ (*** = p<0.001).
Fig 4: Radioresistance in IRP1- cells is associated with iron availability and a free radical-mediated mechanism.Panels a–b: IRP1- and control cells were treated with indicated doses of chemicals and assayed for apoptosis by annexin V & PI staining at 24 h post H2O2 treatment (panel a) or 48 h post staurosporine treatment (panel b). Data were fitted to a non-linear variable slope sigmoidal response curve. Panels c–d: cells were treated with 10 mM Tempol for 15 m at 37°C before exposure to indicated doses of gamma rays. Cells were immediately washed and cultured in regular medium for 48 h then assayed for apoptosis by annexin V & PI content. Two-way repeated measures ANOVA with Bonferroni post-tests was used to measure statistical significance at each dose (** = p<0.01). Panel e: cells were treated with 1 mg/mL of purified human apo-transferrin (control) or transferrin for 6 h in serum-free medium and exposed to equitoxic doses of gamma rays (doses were terminated at 7.5 Gy for control cells because cells were 80% dead or more beyond this dose). Cells were immediately washed and placed in regular culture medium and assayed for apoptosis at 48 h by annexin V & PI content. Data were normalized to viability from apo-tansferrin treated cells and analyzed for significance at equitoxic IR doses using a two-way repeated measures ANOVA with Bonferroni post-tests (* = p<0.05). All data are mean +/− SEM from three independent experiments.
Fig 5: Hydrogen peroxide-induced ROS formation is decreased in IRP1- cells.Panel a: cells were loaded with 100 nM H2-DCFDA and then assayed for increases in fluorescence by flow cytometry following indicated doses of hydrogen peroxide, plotted as fold increase over control. Data are mean +/− SEM of three independent experiments. Two-way ANOVA was used to test significance at each dose (* = p<0.05). Panel b: Representative histograms treated with 0 and 100 µM hydrogen peroxide.
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