EGFRvIII expression maintains stemness of GSCs.

<p>(A) Semiquantitative RT-PCR of EGFR-WT and EGFRvIII in various GSCs (EGFRvIII positive cells; CSC2, X01, X03, X04, X06, X08, X09, MD30, 1123NS, 83NS, and EGFRvIII negative cells; X02, Ex Vivo, and 528NS) and EGFRvIII-overexpressing Astrocyte (Astrocyte-EGFRvIII is used as EGFRvIII positive control). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Immunoblot (IB) analysis of EGFR, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 and X01 cells (day 0) and 10% serum-cultured CSC2 and X01 cells. Serum-cultured CSC2 and X01 cells were harvested after indicated time (days 4, 7, and 9). α-tubulin was used as a loading control. (C, D) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 cells (day 0) and 10% serum-cultured CSC2 cells (day 7) (C) and in serum-free GSC cultured X01 cells (day 0) and 10% serum-cultured X01 cells (day 7) (D). (E) Immunocytochemistry (ICC) of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 and X01 cells that were incubated in serum-free (day 0) or serum medium for 7 d (day 7). Nuclei were counterstained with DAPI (blue). (F) IB analysis of phosphorylated EGFR (p-EGFR), EGFR, Sox2, Nestin, and GFAP in CSC2 cells transfected with EGFRvIII small interfering RNA (siRNA) or its control. α-tubulin was used as a loading control. (G) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. GAPDH was used as a loading control. (H) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. Nuclei were counterstained with DAPI (blue). (I) Limiting dilution assay (LDA) was performed in CSC2 cells transfected with EGFRvIII siRNA or its control. <i>p</i> = 0.00966. (J) IB analysis of p-EGFR, EGFR, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. α-tubulin was used as a loading control. (K) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. GAPDH was used as a loading control. (L) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. Nuclei were counterstained with DAPI (blue). (M) LDA was performed in X02 infected with EGFRvIII-expressing lentiviral or control construct. <i>p</i> = 0.0000266.