Figure 1

In Vitro Characterizations of MKP-1 over-expressing H441GL NSCLC Cells (A) In vitro validation of MKP-1 over-expression in H441GL cells at transcriptional level. Both dominant negative (H441GL/MKP-1CS) and wildtype MKP-1 (H441GL/MKP-1) constructs were transduced into H441GL cells Strong MKP-1 transcripts were visualized by ethidium bromide stain Empty vector pcDNA31 served as a control and demonstrated no increased amount of MKP-1 transcript in the tranduced host GAPDH served as a loading control (B) Immunoblots showing MKP-1 over-expression reduced MAPK activities H441GL cell lysates were immunoblotted with antibodies specific for MKP-1, phosphorylated p38MAPK, ERK, JNK or α-tubulin Phosphorylated MAPKs including p38MAKP, ERK and JNK activities were down-regulated by wildtype MKP-1 over-expression (C) Cellular proliferation was examined in MKP-1 over-expressing H441GL cells using dye exclusion assay H441GL cells transduced with dominant negative MKP-1 (H441/MKP-1CS) and empty vector (H441GL/pcDNA31) exhibited similar proliferation rate whereas wildtype MKP-1 over-expressing H441GL cells (H441GL/MKP-1) demonstrated markedly reduced proliferation rate Data was shown as mean ± SD of three independent experiments (D) Induction of MKP-1 triggered mesenchymal-to-epithelial status switch in H441GL cells by increasing E-cadherin (E-cad) while decreasing Vimentin expression Internal controls used in PCR and Western analyses were GAPDH and α-tubulin respectively.