Lentivirus-based MAPK/ERK pathway activation abolished the effect of OA on EMT of glioma cells.

<p>(<b>A</b>) 10 MOI of Lv-MEK or Lv-GFP was added to the cultures of U-87 MG and GT-1^# as well as 25 µg/mL (50 µM) of OA. 48 h later, total RNA was extracted from the cells and expression level of MEK mRNA was determined by qPCR. The experiments were performed for three times. <i>GAPDH</i> was selected as endogenous control. Their relative expression levels in OA-treated cells to untreated ones were shown as mean ± SD. (<b>B</b>) Under the same conditions, MAPK/ERK-responsive luciferase plasmids were transfected into U-87 MG and GT-1^# cells and Firefly luciferase activity was normalized by that of Renilla luciferase. The luciferase activity in untreated cells was selected as standards. These experiments were repeated for three times and the values were shown as mean ± SD. (<b>C</b>) Immunoblotting assay was perfermed to detect MEK and ERK proteins as well as their phosphorylated forms were also determined in 10 MOI of Lv-MEK or Lv-GFP-infected U-87 MG and GT-1^# cells under 25 µg/mL (50 µM) of OA treatment and GAPDH was selected as endogenous control. (<b>D</b>) 10 MOI of Lv-MEK or Lv-GFP was used to infect U-87 MG cells with or without 25 µg/mL (50 µM) of OA. 48 h later, transcripts of E-cadherin, N-cadherin, Vimentin and Twist1 were quantified. The experiments were performed for three times. <i>GAPDH</i> was selected as endogenous control. Their relative expression levels in the tested cells to ones treated by 25 µg/mL (50 µM) of OA and 10 MOI of Lv-GFP were shown as log₂ (mean ± SD). (<b>E</b>) E-cadherin, N-cadherin, Vimentin and Twist1 proteins were also determined in the cells treated simultaneously by lentivirus and 25 µg/mL (50 µM) of OA and GAPDH was selected as endogenous control.