Oridonin Modulates EGFR Signaling Pathway

In order to evaluate the effect of oridonin on EGFR pathway proteins and validate the in silico results, varying concentrations of oridonin (IC₅₀/4, IC₅₀/2, IC₅₀, 2xIC₅₀, and 4xIC₅₀), determined after the cytotoxicity test on U87MG.ΔEGFR cell line, were applied in a similar way as described previously (Saeed et al., 2015 (link)). Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).