*3.10. Annexin-V/PI Double Staining*

Apoptotic cell death was evaluated and quantified using an Annexin-V FITC and PI double staining kit (Mebcyto® Apoptosis Kit, MBL, Nagoya, Japan) according to the manufacturer's instructions. In brief, 2 × <sup>10</sup><sup>5</sup> HCT116 cells were seeded in 25 cm<sup>2</sup> flasks. The next day, cells were treated with 30 μg/mL of C3 or **1** for 24 h. Both adherent and floating cells were collected in order to detect early and late apoptotic cells. Treated and untreated cells (control) were harvested by trypsinization, washed and suspended in ice-cold PBS. The washed cell pellets were re-suspended in an ice-cold binding buffer containing FITC-conjugated Annexin-V and PI. Samples were incubated at room temperature for 15 min in the dark before analysis by FACS, managed with FACSDiva software. The Annexin V-FITC-negative/PI negative, which are the normal healthy cells population are represented by quadrants Q3. Annexin V-FITC-positive/PI negative cells, which are defined as early apoptotic cells (Q4), whereas the Annexin V-FITC-positive/PI positive are the cells found in late apoptosis (Q2). The Annexin V-FITC-negative/PI-positive cells (Q1) include the necrotic cells. The percentage distributions of normal, early apoptotic, late apoptotic, and necrotic cells were calculated using FACSDiva software (Becton Dickenson, San Jose, CA, USA).

#### **4. Conclusions**

Cyanobacteria are vastly abundant organisms in various ecological niches, and marine filamentous cyanobacteria are a subset known to produce a treasure trove of natural products. Until challenges are overcome for using molecular biology tools to predict and realize the potential chemical arsenal of filamentous cyanobacteria via their biosynthetic gene clusters, a more complete chemical diversity of these organisms can be studied using large environmental collections. The chemical space of extracts produced from these assemblages is largely affected by external factors, such as the associated microbial consortia and environmental conditions, and thus increases the complexity of studying assemblages for new molecule discovery. Metabolomics-based approaches can be used to unravel the chemical potential of such complex samples, and minimize the rediscovery of previously reported compounds. The South China Sea harbors largely untapped filamentous cyanobacteria biodiversity that may be investigated to yield new pharmaceutical lead molecules. In this study, the investigation of a cf. *Neolyngbya* sp. cyanobacterium that was collected near Wenchang, Hainan, China led to the discovery of wenchangamide A (**1**) and characterization of its new chemical scaffold. Compound **1** was found to be a fast-acting and concentration-dependent inducer of apoptosis in HCT116 human colon cancer cells in vitro. Further untargeted LC-MS/MS-based metabolomics suggested the occurrence of an additional analogue, wenchangamide B, for which a structure has been proposed with high confidence. Bioassay results from the fraction containing this related molecule also showed in vitro apoptotic activity using HCT116 cells, suggesting that the core polypeptide-derived scaffold may be a pharmacophore and that the length of the polyketide chain could be tailoring molecules of this class for variable potency or solubility. The further expansion of this chemical class and structure–activity relationship should be evaluated for natural products anticancer drug discovery and development.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md19070397/s1: 1H, 13C, DEPT135, COSY, TOCSY, HSQC-TOCSY, HSQC, HMBC, and ROESY NMR, UV, and IR for compound **1**. Tabulated NMR data for **1** in pyridine-*d*5. MS/MS spectra for compound **1** and the proposed analogue, wenchangamide B. Chiral HPLC chromatograms of hydrolysates of **1** and authentic standards. In vitro bioassay data. Weblinks for the GNPS metabolomic data used to produce Figures 3 and 9. 16S rRNA gene V3–V4 amplicon used to prepare Figure 2C.

**Author Contributions:** Conceptualization, C.B.N. and T.L.-K.; Methodology, L.D., R.B.-S., N.K., S.L., A.I.; Data Analysis, R.B.-S., D.A., A.I., C.B.N., T.L.-K.; Resources, G.P., A.I., K.S., C.Z., H.L., F.T., C.B.N.; Writing—Original Draft Preparation, L.D., R.B.-S., A.I., C.B.N., T.L.-K.; Writing—Review and Editing, all authors; Project Administration, F.F., C.B.N., T.L.-K.; Funding Acquisition, L.D., C.B.N., S.H., X.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Key Research and Development Program of China, funded through MOST (the Ministry of Science and Technology of China; grant 2018YFC0310900 to X.Y., S.H. and C.B.N.), NSFC (The National Natural Science Foundation of China; grants 81850410553 and 82050410451 to C.B.N.), the National 111 Project of China (D16013), CSC (China Scholarship Council; no. 201908330173 to L.D.), and the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund of Ningbo University.

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

**Data Availability Statement:** The datasets generated for this study can be found in the online supplementary materials. Metabolomics data are archived on the GNPS platform and can be found in the following links: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f62b23918fb24bca9f4a234f3 555df50; https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=0e36af9bc15d4d6c901292d5be8ff32b.

**Acknowledgments:** We thank Meirav Avital Shacham, Head of analytical and chromatography unit, the Faculty of Natural Sciences, University of Haifa, Israel and Sagie Schif-Zuck for the use of FACSCanto II (BD) equipment, Faculty of Natural Sciences, University of Haifa. We also thank Larisa Panz, Mass spectrometry Center, Schulich Faculty of Chemistry, Technion, Israel and Shai Zaid, Department of Marine Biology, The Charney School of Marine Sciences, University of Haifa, Israel for their help in MS data acquisition.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

