Fig. 4.

Clustered microRNAs impede GBM escape from genotoxic stress. a Western blot from whole-cell lysate of G34 expressing either negative control or Cluster 3 transgene 24 h after either 15 μM TMZ or 2 Gy ionizing radiation. b Quantification of proteins from a. Values reported represent mean + SD from two separate experiments. c, d Representative western blots for phospho-H2A-x and H2A-x in G34 GSC after 48 h incubation with 15 μM TMZ and further 48 h after TMZ washout, and relative protein quantification (mean + SD, n = 3) e, f Representative western blot for phospho-H2A-x and H2A-x in G34 GSC 30 min, 12 h and 24 h after 2 Gy irradiation, and relative protein quantification based on three independent experiments (reported values are mean ± SD). g FACS-based analysis of cell death and apoptosis in G34 GSC expressing either single miRs or Cluster 3 transgene, in the presence of either 15 μM TMZ (upper row) for 5 days or 5 days after treatment with 2 Gy of ionizing radiation (lower row). Percent of living cells is reported in each left lower quadrant. 7-AAD 7-amino-actynomicin-D. h Cell count per well of G34 GSCs expressing different microRNAs at different time points and in different genotoxic conditions (Left panel: no additional treatment; Central panel: TMZ; Right panel: radiation). Reported is the mean ± SD from three independent experiments. For all graphs, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Student’s t test, two tails). i Survival curve of nude mice intracranially implanted with 10,000 G34 GSC differentially expressing clustered microRNAs, and with/without 5 days treatment with 20 mg/kg IP TMZ starting at day 7 post implantation (six mice/group). **p < 0.01, Log Rank corrected by Bonferroni analysis. GSC glioblastoma stem-like cell