*4.12. LC-MS/MS Analysis*

Resolved protein samples were analyzed using QTRAP® 6500 LC-MS/MS System (AB Sciex LLC, Framingham, MA, USA). Mass spectrometric data were searched against the NCBI database with a taxonomy restriction to 2019 human proteins (172,061 sequences; 53,783,369 residues, 27 July 2020) using MASCOT V2.0 (Matrix Sciences, London, UK).

#### *4.13. Data Analysis*

A bar diagram was plotted for the cytotoxicity test to visualize the "mean percent of inhibition ± standard error" of cell proliferation using Microsoft® Excel 2019 software. The significance of the lactucin doses was compared with control sample data and calculated by "one-way ANOVA" using "IBM® SPSS® Statistics 26". Significant difference at *p* 0.05, 0.01, 0.001, and 0.0001 was calculated and expressed with different Asterix "\*" numbers on the chart or the data table legend. IC50 was calculated by plotting the Log10 dose on *X*-axis versus the Normalized response on the *Y*-axis in Graph Pad Prism 7. Cell cycle cytometry data were analyzed using ModFit LT™ (Version 5.0.9). Apoptosis compensation calculation and data analysis were done using CytExpert™ (Version 2.3.0.84). Protein concentrations were calculated in Microsoft® Excel 2019. WB bands were quantified using ImageJ® (Version 1.52a) and analyzed using Graph Pad Prism 7. NMR data was analyzed, the structure was predicted using MestReNova™, and the chemical structure was made using ChemDraw® Professional. LC-MS/MS results analyzed in MASCOT were combined with WB markers data, and functional enrichment was performed to predict the involved molecular pathway using DAVID Bioinformatics resources [36,37].

#### **5. Conclusions**

In developing anticancer therapy, discovering natural compounds and their mechanisms are the new frontier. They can concurrently affect multiple cell lines, and are helpful in attending to metastasized cancers, while still possessing molecular specificity towards different oncogenes and tumor suppressors. Herein, we reported cytotoxic, cell cycle inhibitory, and apoptosis-inducing properties of Lactucin in A549 and H2347 cells for the first time. We also narrowed down Lactucin's potential target in the A549 cell as MAPK/ERK pathway. Lactucin exerts its effect by lowering the carbon metabolism necessary for metastasized tumor development. To the best of our knowledge, this study was the first attempt at identifying Lactucin's anticancer mechanism in the lung cancer cell line.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27217358/s1, Figure S1: Functional enrichment of ABPP and WB peptides using DAVID Bioinformatics resources predicted Lactucin's interaction with several "Central carbon metabolism in cancer" pathways enzymes (the chart is from KEGG database); Figure S2: Downregulation of the MEK-ERK signaling pathway in NSCLC leads to upregulation of p53 and DNA repair, which downgrade increased carbon consumption-related enzymes (the chart is from KEGG database, red star *p* < 0.05).

**Author Contributions:** K.M.S.U.I. and Y.T. wrote the first draft and developed the figures and tables. F.X. contributed to the writing and argument development. Y.X. and B.W. jointly made critical revisions and approved the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Agricultural Science and Technology Innovation Program of Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (CAAS-ASTIP-G2022-IFST-07 fund; and the CAMS Innovation Fund for Medical Science (CIFMS), grant number 2021-1-I2M-028.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We are incredibly grateful to Yu Tian for her help in constructing the Lactucin probe. We thank Boting Wen and Yulu Wang for the helpful discussion.

**Conflicts of Interest:** There are no conflict of interest to declare.

**Sample Availability:** Source of the compound Lactucin is described in the materials method section.
