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. 2017 Mar 31;8(22):35639–35655. doi: 10.18632/oncotarget.16767

Figure 3. Development and validation of temozolomide resistant model.

Figure 3

A. Schematic representation depicting generation of U373vIII cells with 150μM TMZ resistance (U373vIIIR). Micrographs in lower panel demonstrate the timeline and development of U373vIIIR cells; Panel B1=confluent cells, B2=150 μM TMZ block, B3= TMZ resistant cells, B4=TMZ resistance cells growing to near confluence B. Immunofluorescence staining for the presence of GFAP was conducted on the U373, U373vIII and U373vIIIR cells followed by the secondary antibodies conjugated with green fluorophore. DAPI was used to stain the nuclei. Representative merged images show the cells expressing GFAP Bar=100 μm C. FACS analysis was done to analyze the cell cycle in U373vIII and U373vIIIR cells when treated with 150μM TMZ D. Western blot analysis of EGFR, EGFRvIII, pEGFR and PDK1 protein expression in U373, U373vIII and U373vIIIR cells. GAPDH was used as a loading control E. Semi-quantitative qRT-PCR analysis for EGFR, EGFRvIII and PDK1 in U373 and its EGFRvIII counterparts F. U373vIII or U373vIIIR cells were cultured with 1mM DCA and cell proliferation was measured by MTT assay G. Cells were stained for apoptosis using TUNEL assay. Results represent the mean ± SD of three experiments performed in triplicate.