**2. Results**

#### *2.1. Preaparation of Bryopsis Plumosa Lectins*

Bryopsis lectin was successfully isolated from the crude extract of Bryopsis plumosa. Combining GalNAc and D-Man affinity chromatography was performed to separate each lectin. Stepwise elution of lectins as different monosaccharides was effective in separating BPL1 and BPL3 (Figure 1). The flow-through fraction contained BPL2, which was successfully isolated using mannose affinity chromatography (Figure 1). The purity of each BPL was more than 95%. All the lectins showed similar hemagglutination activities, as previously reported [22–25].

**Figure 1.** Purification of Bryopsis plumosa lectins (BPLs). M, Molecular weight marker; lane 1, crude extract; lane 2, BPL1; lane 3, BPL2; lane 4, BPL3.

#### *2.2. Cell Viability of Lung Cancer Cells and Non-Cancerous Cells against BPLs*

BPL2 inhibited the viability of lung cancer cell lines, whereas BPL1 and BPL3 did not show the inhibitory activity of the tested cell lines at any concentration (Supplementary Figure S1). Cell viability was dependent on the BPL2 concentration and exposure time. BPL2 activity was most effective in the A549 and H1299 lung cancer cell lines, with cell viability being approximately 40% at 100 μg/mL of BPL2 after exposure for 24 h. However, weak toxicity towards non-cancerous cell lines (MRC5, HEK293T, and HaCaT) was observed at 100 μg/mL (~60–70% viability) (Figure 2A). The increase in the exposure time of BPL2 affected cell viability; cell viability was reduced to approximately 20% after 72 h of exposure (Figure 2B). Based on the cytotoxicity results in non-cancerous cell lines, subsequent experiments were performed at a BPL2 concentration of fewer than 25 μg/mL.

According to the colony-forming assay, BPL2 was effective at low concentrations. All cancer cell lines disappeared after treatment with 20 μg/mL of BPL2. In addition, half of the colonies did not survive at 20 μg/mL BPL2 (Figure 3).

**Figure 3.** Colony-forming assay for the anticancer activity of BPL2. A549, H460, and H1299 cancer cell lines were used for these studies.
