Fig. 3

Inhibition of GSC self-renewing capacity by knockdown of selected defense signatures of GSC-500 μM TMZ. a. GSC-500 μM TMZ were treated with siRNA targeting indicated defense signatures of GSC-500 μM TMZ in the presence or absence of 35 μM TMZ. Representative photos (D431-500 μM TMZ) were taken 7 days after treatment (a). On-target gene knockdown by siRNA treatment was verified by sqRT-PCR analysis 48 h after transfection (b). The graph shows the mean values of mRNA expression levels of siRNA-targeted genes in GSC-500 μM TMZ lines relative to those of treated with negative control siRNA. All values were relative to those of the internal control gene β-actin, with values of GSC-500 μM TMZ representing the percentage of mRNA expression levels relative to that after treatment with negative control siRNA, which was converted to 100 %. Data represent mean values ± SD of triplicate measurements in three independent experiments in two GSC lines (D431-500 μM TMZ, E445-500 μM TMZ). All gene-targeted values represent statistically significant reduction of mRNA levels (P < 0.001). b. Gene knockdown verification on the protein level. The siRNA-mediated knockdown of GAPDH and NNMT expressions in GSC at protein levels was determined by measurement of GAPDH enzymatic activity (a) and Western blot analysis (b), respectively. Data represent mean values ± SD of triplicate measurements in three independent experiments. **p < 0.001 in relation to treatment with negative control siRNA. c. The effects of gene knockdown on GSC self-renewing capacity. The GSC growth under the indicated treatment conditions was determined by MTS assay, which was carried out 72 h after transfection. Data represent mean values ± SD of triplicate measurements in three independent experiments. *p < 0.05 and **p < 0.001 in relation to cells treated with negative control siRNA