*2.4. Lactucin Affects the MAPK Signaling Pathway by Downregulating MEK, ERK Phosphorylation*

Effects of Lactucin on metabolic markers of lung cancer cells were observed through WB, Figure 4A. Protein lysate from 24 h 80 μM Lactucin-treated A549 cells showed a different level of expression of metabolic markers, Figure 4B. Lactucin dose-dependently increased the expression of PTEN, a tumor suppressor protein that negatively regulates the activation of Akt to p-Akt, Figure 4 [26]. Dose-dependent MEK 1/2 and ERK activation inhibition indicate that Lactucin inhibits A549 cell proliferation by downregulating the MAPK/ERK pathway. We used a biotinylated Lactucin probe to confirm this and isolated Lactucin interaction proteins from A549 cells.

**Figure 3.** Effect of Lactucin on apoptosis induction of lung cancer cells. Lactucin and equivalent volume of the vehicle (DMSO) exposure (24 h) induced apoptosis of lung adenocarcinoma A549 (80 μM) and H2347 (70 μM) cells (**A**) were measured by flow cytometry. WB analysis of c-Caspase-3, c-PARP, Bax, and Bcl-2 protein expression was performed after A549 and H2347 cells were exposure to Lactucin at 0, 20, 40, and 80 μM for 24 h (**B**–**D**). Data are expressed as the means ± standard deviation of triplicate experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001 indicate result's significant difference from the control.
