Figure 1.

Sea hare hydrolysate-induced polarization to M1 of RAW264.7 cells. (A) Effect of sea hare hydrolysate (SHH) on cell viability. Different concentrations of SHH were used to treat RAW264.7 cells for 24 h. Each bar is the mean ± standard deviation (SD) obtained from five independent experiments (n = 5). (B) Morphological changes in RAW264.7 cells activated by SHH treatment. Numbers (1, 10, and 100) above the figures represent the concentration (μg/mL). LPS (1 μg/mL) was used as a positive control. Scale bar, 15 μm. (C) SHH-induced increase in iNOS and TNF-α expression. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as a loading control to compare the mRNA expression level among treatments. (D) No expression of Arg-1, a marker of M2, in RAW264.7 cells. LPS (1 μg/mL) was used as a positive control for induction of iNOS and TNF-α expression. (E) No effect of IL-4 and SHH on Arg-1 expression in RAW264.7 cells. The Arg-1 was expressed in the IL-4-treated mouse peritoneal macrophages. (F) Increase in the phagocytic ability of RAW264.7 cells by SHH treatment. Cells cultured in 96-well black plates were treated with LPS or SHH and loaded with latex bead rabbit IgG FITC complex. The degree of phagocytosis was analyzed using a fluorescence microplate reader. Each bar is the mean ± SD obtained from nine independent experiments (n = 9). * p < 0.05 compared to control (CTL). FI represents fluorescence intensity. (G) RAW264.7 cells phagocytized A549 lung cancer cells. The cancer cells were transfected with green fluorescent protein (GFP) and co-cultured with RAW264.7 cells for 24 h under SHH treatment. Strong green fluorescence instead of dots shows A549 cells transfected with GFP. The bar graph shows the percentages of GFP positive cells (RAW264.7 cells that phagocytized A549 cells). Each bar is the mean ± SD obtained from four independent experiments (n = 4). LPS treatment was used as a positive control. NS, not significant. Scale bar, 30 μm. * p < 0.05 compared to control (CTL).