*4.1. Preparation of Bryopsis plumosa Lectins (BPLs)*

*Bryopsis plumosa* cultured in our laboratory was used to extract BPLs. BPLs were isolated following a previously reported method by Han et al. [23–25]. *Bryopsis plumosa* was harvested and washed with 1× Tris-buffered saline (TBS, pH 7.5) containing 1 mM CaCl2 and 1 mM MgCl2. The harvested samples were ground into a fine powder, after exposure to liquid nitrogen, using a mortar and pestle. Five volumes of ice-cold 1× TBS were added to the ground samples and incubated for 3 h at 4 ◦C. The incubated sample was centrifuged at 25,000× *g* for 30 min at 4 ◦C, and the cell debris was removed. The crude extract was loaded directly onto an affinity chromatography column. A Bio-Rad NGC FPLC system (Bio-Rad Laboratories, Hercules, CA, USA) was used for chromatographic analysis at a flow rate of 1 mL/min. First, GalNAc-agarose was used to purify BPL1, BPL3, and BPL4 using a stepwise elution method. The affinity column was washed with 10 volumes of 1× TBS. BPL3 and BPL4 were eluted using 0.2 M of GlcNAc in 1× TBS and then BPL1 was eluted using 50 mM GalNAc in 1× TBS. The flow-through from GalNAc-agarose, which contained mannose-binding lectin, was loaded onto the mannose-agarose. BPL2 was eluted with 0.5 M D-mannose dissolved in 1× TBS.

All lectins were confirmed using SDS-PAGE and UV spectrophotometry. The BPLs were dialyzed using 1× PBS and stored at −20 ◦C until use.

#### *4.2. Cell Culture and Viability Assay (Determination of Viability of Tumo Cells)*

Metastatic lung cancer cell lines (A549, H460, and H1299) were used to determine tumor cell viability. Non-cancerous and immortalized cells (MRC5, HEK293T, and HaCaT cells) were used as controls (Supplementary Table S1). All cells used for the cell viability test were cultured at 37 ◦C under atmospheric conditions of 5% CO2 in fetal bovine serum (FBS) containing antibiotics (penicillin and streptomycin). The growth rate of the cells was determined by CCK-8 analysis. Aliquots of each cell line were added to 96-well plates to achieve a cell number of 5 × <sup>10</sup><sup>3</sup> cells per well and incubated in a 5% CO2 atmosphere at 37 ◦C for 24 h. BPLs (BPL1, 2, and 3) at concentrations of 25, 50, and 100 μg/ml, respectively, were added to the cells and then incubated for 24–72 h. After incubation, the culture medium was removed and the cells were incubated in a fresh culture medium containing CCK-8 solution for 3 h. Cell viability was determined by measuring absorbance at 450 nm using a 96-well plate reader (Spectramax i3x; Molecular Devices, San Jose, CA, USA). Live cells were calculated as a percentage. All experiments were repeated at least three times.

#### *4.3. Determination of Cell Growth Rate Based on BPL Treatments*

The cell lines were cultured in the same manner as for the cell viability test. A colonyforming test was performed to compare the growth rate among cell lines treated with different concentrations of BPLs. Each cell line was divided into 1 × <sup>10</sup><sup>3</sup> cells per 30 mm dish and cultured at 37 ◦C in a CO2 incubator for 24 h. The cell lines were treated with

BPLs at concentrations of 10 and 20 μg/mL. After being cultured for 7 days, the culture medium was discarded and stained with 0.5% crystal violet solution for 10 min. The stained cells were washed several times with 1× PBS and the growth rate was observed under a microscope.

#### *4.4. Determination of Migration and Invasion of Cancer Cells*

The migration and invasion assays were performed using a Transwell (Falcon, BD labware, Bedford, MA, USA) with a 0.8 μm pore size. The EMT protein marker was used to analyze the migration ability. The lung cancer cell lines were incubated at 37 ◦C for 48 h after inoculation into the migration well to obtain a density of 1 × <sup>10</sup><sup>5</sup> cells/well, which were stained using crystal violet solution.

#### *4.5. Comparison of the Expression Level of Cell Migration and Invasion-Related Genes, and EGFR-Related Proteins*

The expression levels of cancer cell-related genes (ZEB1, vimentin, and Twist) were analyzed using RT-PCR. Total RNA from each cell was isolated using the TRIzol RNA extraction solution. RNA quality was determined by gel electrophoresis using a UVspectrophotometer. First-strand cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Penzberg, Germany). One microgram of total RNA was used for first-strand cDNA synthesis. Primer information for the RT-PCR is listed in Supplementary Table S2. Amplification was performed using an Applied Science PCR machine under the following conditions: pre-denaturation at 94 ◦C for 5 min, 30 cycles of denaturation at 94 ◦C for 5 min, annealing at 56 ◦C for 1 min, extension at 72 ◦C for 1.5 min; and final extension at 72 ◦C for 5 min. The relative expression levels of target genes were analyzed by gel electrophoresis.

For analysis of EMT-related protein levels, Western blot analysis was performed. Cells were lysed in a buffer with protease inhibitor cocktails (Sigma-Aldrich). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto NC membranes. Membranes were incubated with each antibody in a blocking solution overnight. After washing with Tris-buffered saline, membranes were incubated with mouse secondary antibody (Abcam, Cambridge, MA, USA) and visualized using a Supersignal west atto ultimate sensitivity substrate (Thermo Scientific, A38555). Antibodies specific for N-cadherin (59987), ZEB1 (515797), Vimentin (6260), Snai1 (271977), and β-actin (47778) were purchased from Santa Cruz Biotechnology.

The analysis of EGFR-related proteins was performed by the same procedure as for EMT-related proteins. The antibodies for EGFR (377547), pERK (7383), ERK (514302), AKT (5298), and pAKT (271966) were obtained from Santa Cruz Biotechnology.

#### *4.6. The Effect of Concurrent Treatment (Gefitinib and BPL2) on Lung Cancer Cell Viability*

Human cancer cell lines (A549 and H460) were used to determine the effect of concomitant drug administration. Cell lines were prepared following the method described above for cell viability. Concomitant gefitinib-BPL2 or BPL2 (10 μg/mL) and gefitinib (10 μM) monotherapy were used to treat the cell lines. The treated cancer cell lines were incubated for 24 or 48 h, and viability was measured using CCK-8 analysis kits.

#### *4.7. Flow Cytometric Analysis for Apoptosis in BPL2-Treated Lung Cancer Cells*

A549 and H1299 cell lines were treated with 20 μg/mL of BPL2 and incubated for 48 h in a CO2 incubator. The cells were double stained using the AnnexinV/PI apoptosis detection kit (556547; BD Biosciences, San Jose, CA, UAS) following the manufacturer's instructions. Apoptosis was determined using a flow cytometer (Accuri C6 Plus; BD Biosciences.

### **5. Conclusions**

The anticancer activity of algal lectins has been studied for several decades. Lectins from *Bryopsis plumosa* have been suggested as candidate antitumor agents. Herein, the anticancer activity of BPL2 was demonstrated in lung cancer cell lines, and the inhibition of cell migration and invasion by BPL2 was presumed to be related to the EMT pathway. Concurrent treatment with BPL2 and gefitinib had a synergetic effect on investigated lung cancer cell lines. Therefore, BPL2 could be a good candidate anticancer agent for lung cancer therapy.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md20120776/s1: Table S1: Cancer and non-cancerous cell lines used for the anticancer assay, Table S2: Primer sequences used for RT-PCR analyses. Figure S1: Effect of BPL on the viability of A549 and H460 cell lines using MTT assays.

**Author Contributions:** Conceptualization, J.W.H.; experimental design, J.W.H. and J.H.L.; experiments, J.W.H., J.H.L., S.B.L., H.K. and J.M.S.; data analysis, J.W.H. and J.H.L.; manuscript writing, J.W.H., M.Y., H.S.A. and J.H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Marine Biodiversity Institute of Korea (No. 2022M00400).

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

#### **References**

