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Figure 5: Knockdown of PTEN and overexpression of EGFR cooperate in vitro to oncogenically transform immortalized human Schwann cells. (a) PiggyBac (PB) constructs used to knock down PTEN (PB-shPTEN) and/or overexpress EGFR (PB-EGFR) in HSC2λ immortalized human Schwann cells. CAG: cytomegalovirus early enhancer element and chicken beta-actin promoter; PGK: phosphoglycerate kinase; EEF1A1: eukaryotic translation elongation factor 1 alpha 1 promoter; IRES: internal ribosome entry site; Gfp: green fluorescent protein; pA: polyadenylation signal; Puro: puromycin resistance gene; triangles: PB-specific inverted terminal repeat sequences. Quantitative PCR analysis demonstrating that PTEN mRNA levels are reduced (b) and EGFR mRNA levels are increased (c) when these constructs are stably transfected into HSC2λ cells. (d) MTS proliferation assay shows that PTEN knockdown or EGFR overexpression alone do not change the rate of proliferation compared to control transfected cells, but when combined significantly increase cellular proliferation. (e) Soft agar colony formation assay demonstrates that PTEN knockdown moderately increases colony formation, but in the context of EGFR overexpression, reduction in PTEN significantly increases the number of colonies formed. * and ** , unpaired t-test; mean ± standard deviation.