Fig 1.
Analysis of EGFR amplification by 2C CISH and qPCR and corresponding EGFR protein expression by immunohistochemistry in GBM PDX tissue of HROG22, HROG33 and HROG59.
A) 2C CISH of HROG22 (400x magnification), B) EGFR IHC of HROG22 (200x magnification), C) 2C CISH of HROG33 (400x magnification), D) EGFR IHC of HROG33 (200x magnification), E) 2C CISH of HROG59 (400x magnification), F) EGFR IHC of HROG59 (200x magnification); red signals: centromere of chr. 7; green signals: EGFR in the 2C CISH analyses; G) qPCR analysis of GBM PDX tissue, error bars represent the standard deviation of triplicate analyses.
Fig 2.
EGFR amplification is maintained over several in vitro passages, but lost in the 10% FCS control and with high EGF concentrations.
qPCR analysis of A) xHROG22, B) xHROG33, C) x HROG59 cell lines grown under all culture conditions at passage 2 (grey bars), passage 6 (dark grey bars) and passage 10 (light grey bars). Black bars represent the respective PDX prior to cell culture establishment. D) qPCR analysis of the 10% FCS control (white bars) and the 30 ng/ml EGF cell line (grey bars) of xHROG33 over several in vitro passages. Error bars represent the standard deviation of triplicate analyses. * p<0.05, Tukey test.
Fig 3.
2C CISH analysis confirms qPCR data of EGFR amplification.
2C CISH analyses of xHROG22, xHROG33 and xHROG59 cell lines established under different conditions as indicated at passage 10 of cell culture; 400x magnification.
Fig 4.
EGFR protein expression is concordant with genomic EGFR amplification.
EGFR immunohistochemistry staining of paraffin embedded xHROG22, xHROG33 and xHROG59 cells grown under different conditions as indicated at passage 10 of cell culture; 200x magnification.
Fig 5.
EGF withdrawal restores EGFR amplification.
A and D) qPCR analysis of xHROG33 (A) and xHROG59 (D) cultured with 30 ng/ml EGF until passage 10 (grey bars) and without EGF for 5 additional passages (dashed bars), respectively. Error bars represent the standard deviation of triplicate analyses; B and E) 2C CISH analysis of paraffin embedded samples of xHROG33 (B) and xHROG59 (E) after 5 passages post EGF withdrawal (P15), C and F) EGFR immunohistochemistry staining of paraffin embedded samples of xHROG33 (C) and xHROG59 (F) after 5 passages post EGF withdrawal (P15). 400x magnification. * p<0.05, Tukey test.