Journal: Journal of Radiation Research

Article Title: Inhibition of mitochondrial RNA polymerase sensitizes cancer cells to radiation by inhibiting mitochondrial respiration

doi: 10.1093/jrr/rrag021

Figure Lengend Snippet: Effect of the POLRMT inhibitor IMT1 on radiation-induced apoptosis. (A, B) Apoptosis was measured by Annexin V/PI flow cytometry in A549 cells pretreated for 2 days with DMSO or 0.5 μM IMT1, irradiated with 5 Gy or left un-irradiated (0 Gy), then incubated under the same conditions and analyzed 5 days post-irradiation. One representative plot of three independent experiments is shown in (A). Apoptosis was defined as the sum of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells (B). Data and bars represent the mean ± SD for three independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test; ** P < 0.01, *** P < 0.001. n.s., not significant. (C) Western blot of BCL-2 (upper panel), BAX (middle panel) and β-actin as a loading control (lower panel) in untreated A549 cells, A549 cells treated with 0.5 μM IMT1 alone, A549 cells irradiated with 5 Gy without addition of IMT1, and A549 cells treated with 0.5 μM IMT1 plus X-ray irradiation (5 Gy), collected at the same time points as the those in Annexin V/PI assay (A and B).

Article Snippet: X-ray irradiation was performed using a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA) at 130 kV, 5 mA, with a dose rate of 1.359 Gy/min with a 0.5 mm aluminum filter.

Techniques: Flow Cytometry, Irradiation, Incubation, Western Blot, Control

Journal: Journal of Radiation Research

Article Title: Inhibition of mitochondrial RNA polymerase sensitizes cancer cells to radiation by inhibiting mitochondrial respiration

doi: 10.1093/jrr/rrag021

Figure Lengend Snippet: Time course of γH2AX and RAD51 foci formation after X-irradiation in A549 cells treated or untreated with the POLRMT inhibitor IMT1. (A) Representative immunofluorescence images of γH2AX foci (red) in A549 cells treated with DMSO (upper panel) and those treated with 0.5 μM IMT1 (lower panel), in an un-irradiated state (0 Gy) and at 1, 8 and 24 h after 1 Gy X-ray irradiation. Scale bar, 20 μm. (B) Percentages of cells containing more than five large γH2AX foci in A549 cells treated with DMSO or 0.5 μM IMT1, in an un-irradiated state (0 Gy) and at 1, 8 and 24 h after 1 Gy X-ray irradiation. (C) Representative immunofluorescence images of RAD51 foci (green) in A549 cells treated with DMSO (upper panel) and those treated with 0.5 μM IMT1 (lower panel), in an un-irradiated state (0 Gy) and at 2, 8 and 24 h after 10 Gy X-ray irradiation. Scale bar, 20 μm. (D) Percentages of cells containing more than five large RAD51 foci in A549 cells treated with DMSO or 0.5 μM IMT1, in an un-irradiated state (0 Gy) and at 2, 8 and 24 h after 10 Gy X-ray irradiation. In (B) and (D), a total of 200 cells were examined for each cell clone and at each time point. Columns and bars represent the mean and SD of three independent experiments, respectively. Statistical significance was determined using Student’s t -test. n.s., not significant.

Article Snippet: X-ray irradiation was performed using a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA) at 130 kV, 5 mA, with a dose rate of 1.359 Gy/min with a 0.5 mm aluminum filter.

Techniques: Irradiation, Immunofluorescence

Journal: Journal of Radiation Research

Article Title: Inhibition of mitochondrial RNA polymerase sensitizes cancer cells to radiation by inhibiting mitochondrial respiration

doi: 10.1093/jrr/rrag021

Figure Lengend Snippet: Time course analysis of the effect of the POLRMT inhibitor IMT1 on cell-cycle progression after X-irradiation. (A) Representative cell cycle distribution histograms of A549 cells treated with DMSO or 0.5 μM IMT1 in un-irradiated steady states (0 Gy) at the indicated time points (2 upper panels) and those at 0, 12 and 24 h after 5 Gy X-ray irradiation (2 lower panels). (B) The proportion of cells in different phases in the cell cycle (G1 phase, S phase and G2/M phase) are shown as stacked bars for A549 cells treated with DMSO or 0.5 μM IMT1 in un-irradiated steady states (0 Gy) at the indicated time points (upper panel) and for those at 0, 12 and 24 h after 5 Gy X-ray irradiation (lower panel). Data represent the mean and SD of three independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. In each condition and each time point in the analyses, there were no significant differences in the proportion of cells in G1 phase, S phase and G2/M phase between IMT1-treated and untreated cells.

Article Snippet: X-ray irradiation was performed using a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA) at 130 kV, 5 mA, with a dose rate of 1.359 Gy/min with a 0.5 mm aluminum filter.

Techniques: Irradiation

Journal: Journal of Radiation Research

Article Title: Inhibition of mitochondrial RNA polymerase sensitizes cancer cells to radiation by inhibiting mitochondrial respiration

doi: 10.1093/jrr/rrag021

Figure Lengend Snippet: Effects of the POLRMT inhibitor IMT1 in combination with radiation on mitochondrial respiration and rescue effects of supplementation with galactose on radiosensitivity. (A) Schematic diagram illustrating the typical measurement of OCR using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies), depicting key mitochondrial respiration parameters and sequential injections of mitochondrial inhibitors (downward arrows). (B) Diagram of the mitochondrial respiratory chain (complexes I–IV) and ATP synthase (complex V). Targets of all mitochondrial inhibitors used in the Seahorse XF Cell Mito Stress Test Kit are indicated. (C) Representative OCR trace for untreated A549 cells, cells treated with radiation alone (X-ray (5 Gy)), and cells treated with IMT1 plus radiation (X-ray (5 Gy)). Three independent experiments were performed. (D) Quantified mitochondrial maximal respiration capacity in untreated A549 cells, cells treated with radiation alone (X-ray (5 Gy)), and cells treated with 0.5 μM IMT1 plus radiation (X-ray (5 Gy)). Each data point represents a mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test; ** P < 0.01, *** P < 0.001. (E) Time course of experimental procedures for testing whether replacing glucose in cell culture medium with galactose can rescue radiosensitivity. After culturing the cells for more than 3 weeks in regular culture medium containing 25 mM glucose or in no glucose medium with 10 mM galactose, IMT1 was added. Two days later, the cells were either X-irradiated or un-irradiated and subjected to the colony formation assay. (F and G) Survival of the A549 (F) and HeLa (G) cells treated with 0.1 μM IMT1 plus X-irradiation (3Gy) in glucose medium or galactose medium, which was normalized to survival of un-irradiated control. Columns and bars represent the mean and SD of three independent experiments, respectively. Statistical significance was determined using Student’s t -test; *** P < 0.001.

Article Snippet: X-ray irradiation was performed using a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA) at 130 kV, 5 mA, with a dose rate of 1.359 Gy/min with a 0.5 mm aluminum filter.

Techniques: Cell Culture, Irradiation, Colony Assay, Control