*2.1. Lactucin Inhibits Lung Adenocarcinoma Cells Proliferation*

The effect of incrementing concentrations of Lactucin on normal lung cells and lung adenocarcinoma cells upon 24 h exposures was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compared to respective control groups, higher Lactucin doses significantly inhibit A549 (IC50 = 79.87 μM) and H2347 (IC50 = 68.85 μM) lung adenocarcinoma cells' proliferation in a dose-dependent manner while not affecting MRC-5 cells' proliferation, Figure 1A. However, at different exposure periods (12, 24, and 28 h), Lactucin showed significant inhibitory activity on A549 cell proliferation at 24 h and 48 h exposures at higher doses only, Figure 1B. Given that Lactucin can significantly inhibit lung adenocarcinoma cell proliferation at 24 h, exposure at a higher amount without affecting normal lung cells, we used respective IC50 concentrations of Lactucin for treating A549 and H2347 cells in further cytological experiments. A549 and H2347 cells treated with incrementing doses of Lactucin for 24 h showed a significant dose-dependent increase in the expression of LC3-II (17 kDa) in H2347 cells; and LC3-I (19 kDa) in A549 and H2347 cells, Figure 1C,D.

**Figure 1.** Lactucin significantly inhibits the proliferation of A549 and H2347 lung adenocarcinoma, while it doesn't inhibit MRC-5 normal lung cell growth in a dose (**A**) and time (**B**) dependent manner. Lactucin significantly induces dose-dependent expression of LC3-II in H2347 cells (**C**,**D**), detected by WB analysis. Here \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001 indicate significantly different from control.

#### *2.2. Effects of Lactucin on Cell Cycle Progression in Lung Adenocarcinoma Cells*

Since Lactucin inhibited lung cancer cell proliferation, its cell cycle inhibitory property and related markers were analyzed. Cytometric analysis of DNA Content of 24 h lactucin treated (with respective IC50 concentrations) lung adenocarcinoma cells A549 and H2347 were performed. It revealed that Lactucin inhibits the cell cycle at the G0/G1 phase, Figure 2A. It was further supported by the western blot (WB) analysis of Lactucintreated (24 h) adenocarcinoma (A549 and H2347) cell's protein, Figure 2B. Both cyclins (cyclin B1 and cyclin D1) and cyclin-dependent protein kinases (CDKs) (CDK2 and CDK4), which regulate the cell cycle through mutual interactions, were dose-dependently downregulated, Figure 2C,D. However, p21 and p53, the two CDKs inhibitors we tested, were dose-dependently upregulated. This result suggests Lactucin inhibits the cell cycle of lung adenocarcinoma cells at the G0/G1 phase by upregulating CDKs inhibitor p21 and p53, downregulating cyclins and CDKs expression.

#### *2.3. Effects of Lactucin on Apoptosis in Lung Adenocarcinoma Cells*

The apoptosis-inducing capacity of Lactucin was measured by flow cytometry analysis of treated A549 and H2347 cells. As shown in Figure 3A, after 24 h incubation with respective IC50 concentrations of Lactucin, A549 cell's apoptosis increased from 1.93% to 13.42%, and H2347 cell's apoptosis rose from 1.42% to 40.70%. We also observed significant induction of early apoptosis in A549 (from 3.86% to 18.35%) and H2347 (6.07% to 17.36%) cells, Figure 3A. Apoptosis-related indicators such as Bax, Bcl-2 expression; and Caspase-3, and PARP activation, were also compared through WB, Figure 3B. We observed that Lactucin induced a dose-dependent increase in the concentration of both peptides of cleaved Caspase-3, Figure 3C, cleavage of PARP, expression of Bax, and inhibited the expression of Bcl-2, Figure 3D. The upregulation of Bax, downregulation of Bcl-2, and cleavage induction of Caspase-3 and PARP indicate that Lactucin has apoptosis-inducing properties.

**Figure 2.** Lactucin and equivalent volume of vehicle (DMSO) exposure (24 h) induced cell cycle inhibition of lung adenocarcinoma A549 (80 μM), and H2347 (70 μM) cells (**A**) were measured by flow cytometry. WB analysis of cyclin B1, cyclin D1, CDK2, CDK4, p21, and p27 protein expression in A549 and H2347 cells after exposure to Lactucin at 0, 20, 40, and 80 μM for 24 h (**B**). Lactucin significantly downregulates Cyclin B1 and Cyclin D1 (**C**), CDK4, and CDK2 (**D**) and downregulates the p21 and p53 (**E**) expression in both A549 and H2347 cells in a dose-dependent manner. Data are expressed as the means ± standard deviation of triplicate experiments. \* *p* < 0.05, \*\* *p*< 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001 were considered significant.
