U87R cell line is radioresistant.

<p>(A, B) U87R cells has more foci number than U87 cells. Indicated cells were exposed to several radiation dose and the experiments were repeated three times. The numbers of foci formation were presented as bar graphs. *p<0.05, **p<0.01. (C) U87R cells have more foci fraction than U87 cells. Foci fractions were calculated by dividing the number of colonies formed after radiation by the corresponding number of colonies formed without radiation from experiments in (A and B). (D) U87R cells get less effect than U87 cells from radiation on growth pattern. U87R and U87 cells plated in 96-well culture plates were exposed to radiation with 3 Gy cell growth was monitored by measuring cell numbers with MTS assay. **p<0.01.</p

The association of miR-135b or GSK3β expression with IR resistance is clinically relevant.

<p>(A) The GSK3β protein levels of were detected in a cohort of 30 GBM tissues and 30 normal brain tissues using immunohistochemistry assays. (B) Quantitation of GSK3β expression in 30 glioblastoma multiforme (GBM) samples and 30 normal brain tissues (Normal). GAPDH served as the loading control. Alteration of expression is shown as box plot presentations. **p<0.01. (C) Quantitation of miR-135b or GSK3β expression in 30 glioblastoma multiforme (GBM) samples and 30 normal brain tissues (Normal). GAPDH served as the loading control. Alteration of expression is shown as box plot presentations. **p<0.01. (D) The correlation analysis showed that the expression of miR-135b was negatively correlated with the expression of GSK3β proteins in normal brain and GBM tissues (p = 0.07). (E, F)The relative expression level of miR-135b or GSK3β was determined by qRT-PCR in ten paired samples of primary and recurrent GBM. *p<0.05.</p

miR-135b targets GSK3β.

<p>(A) Schematic of predicted miR-135b site in the 3′UTR of GSK3β mRNA, which broadly conserved among vertebrates. (B) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level and mRNA level by qRT-PCR and Western blot. (C) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level by immunofluorescence. (D) miR-135b expression interference negatively regulated GSK3β protein expression by Western blot. (E) miR-135b expression interference negatively regulated GSK3β protein expression by immunofluorescence. (F) Luciferase reporter assays were performed after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-135, or relevant scramble controls. Data shown were the mean±SD of six replicates and were representative of three independent experiments. *p<0.05.</p

GSK3β overexpression reverses radioresistance of U87R cells.

<p>(A) Transfection of GSK3β vector increased GSK3β protein expression. (B–C) GSK3β ovexpression reversed radioresistance of U87R cells detected by foci formation assay. *p<0.05.</p