Fig 2: UW228 cells show increased radiation resistance and increased ABC transporter expression after long-term weekly radiation exposure. (A) Enhanced radiation resistance of cells exposed repeatedly to 3 Gy, by resazurin based total viability assay. Cells were assessed for resistance 2.5 weeks after receiving their final treatment dose. Mean survival of 4 long-term independently cultured sub-lines for each treatment shown. Error bars +/- 1 SD, “***” indicates p-value < 0.001. (B) Elevated expression of ABCA1 and (C)ABCG2, in the independent sub-lines, by qRT-PCR. Points represent relative gene expression for each line derived from the mean ?Ct of quadruplicate multiplex assays. Horizontal bars show the geometric mean for all sub-lines (“*” indicates p-value < 0.05, “**” indicates p-value < 0.01).

Fig 3: UW228 medulloblastoma cells surviving 10 Gy radiation show elevated expression of c-Myc and several ABC transporters. Pair-matched live non-apoptotic cell populations, from 0 Gy (control) and 10 Gy treated (surviving) cells in multiple independent experiments, were isolated using FACS. Analysis by qRT-PCR showed (A)c-Myc, (B)ABCG2 and (D)ABCA1 are elevated in surviving cells. Bars show relative gene expression derived from the mean ?Ct of quadruplicate multiplex assays. To clearly display variation between experiments (both in terms of magnitudes of expression and the difference between control and resistant cells), independent experiments are shown individually. The fold-change value for each pair-matched experiment is also indicated (being the relative expression for surviving cells divided by that for control cells, or 2-??Ct). Error bars (asymmetric on gene expression scale) represent symmetric +/- 1 SD in the Ct space. When surviving cells are compared with control cells over all pooled biological experiments, p-values are <0.05 for all three genes. (C)ABCG2 was also elevated at the protein level when assessed by flow cytometry.

Fig 4: Daoy Medulloblastoma cells surviving 10 Gy radiation show elevated expression of several ABC transporters. Pair-matched live non-apoptotic cell populations, from 0 Gy (control) and 10 Gy treated (surviving) cells in multiple independent experiments, were isolated using FACS. Analysis by qRT-PCR shows (A)ABCG2 and (B)ABCA1 are elevated in surviving cells. Bars show relative gene expression derived from the mean ?Ct of quadruplicate multiplex assays. Independent experiments are shown individually, to clearly display variation between experiments (both in terms of magnitudes of expression and the difference between control and resistant cells). The fold-change value for each pair-matched experiment is also indicated (being the relative expression for surviving cells divided by that for control cells, or 2-??Ct). Error bars (asymmetric on gene expression scale) represent symmetric +/- 1 SD in the Ct space. When surviving cells are compared with control cells over all pooled biological experiments, p-values are <0.05 for both genes.