Figure 2

HMGA1 overexpression or silencing modulate the sensitivity to cetuximab and 5-fluorouracil. (A) GEO cells stably transfected with a construct expressing the HMGA1 gene (GEO-HMGA1) or with empty vector (GEO pCEFL-HA) and analyzed by western blot using HMGA1 and β-actin antibodies. β-Actin was used as loading control (Left Panel). qRT-PCR analysis of HMGA1 mRNA in GEO pCEFL-HA and GEO-HMGA1 cells. Relative expression values indicate the relative change in HMGA1 mRNA expression levels between GEO pCEFL-HA and GEO-HMGA1 and normalized with β-actin (Right Panel). The error bars represent the mean ± S.D. of three independent experiments performed in triplicate. (B) GEO CR stably transfected with a short hairpin RNA targeting the HMGA1 gene (GEO CR shHMGA1) or the empty vector (GEO CR pLKO.1) and analyzed by western blot using HMGA1 and β-actin antibodies. β-Actin was used as loading control (Left Panel). qRT-PCR analysis of HMGA1 mRNA in GEO CR pLKO.1 and GEO CR shHMGA1. Relative expression values indicate the relative change in HMGA1 mRNA expression levels between GEO CR pLKO.1 and GEO CR shHMGA1, normalized with β-actin, assuming that the mean value of GEO CR pLKO.1 was equal to 1 (Right Panel). The error bars represent the mean ± S.D. of three independent experiments performed in triplicate. (C-D) Cell viability assay of GEO pCEFL-HA and GEO-HMGA1 (C) and GEO CR pLKO.1 and GEO CR shHMGA1 (D) cells treated with increasing doses of cetuximab or 5-fluorouracil. Values are the mean ± S.D. of three experiments performed in triplicate.The curves were fitted using nonlinear regression (Graph Pad Prism 5.0. program). The concentration is shown as logarithmic function. *p < 0.05 versus empty vector-transfected cells (GEO pCEFL-HA or GEO CR pLKO.1). (E-F) IC50, inhibitory concentration that kills 50% of cell population and Resistance index calculated as IC50 GEO-HMGA1 /IC50 GEO pCEFL-HA cell line (E) or IC50 GEO CR pLKO.1/ IC50 GEO CR shHMGA1.