Journal: Molecules

Article Title: A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells’ Proliferation

doi: 10.3390/molecules23123202

Figure Lengend Snippet: Peptide B11 affects the cell morphology and induces the apoptosis of HeLa cells. ( A ) Changes in cell morphology following treatment with PBS, peptide B11, and 5-FU for 24 h. Micrographs were obtained using an inverted microscope (20×). ( B ) Changes in cell nuclei morphology following treatment with PBS, peptide B11, and 5-FC for 24 h. The 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stained nuclei were observed with a fluorescence microscope (20×). ( C ) Flow cytometric analysis of apoptosis in HeLa cells after 8 h to 48 h of treatment with PBS, peptide B11, and 5-FU, and staining with Annexin V/propidium iodide (Annexin V/PI). Quadrants: lower-left represent live cells (Annexin V negative/PI negative); lower-right represent early apoptotic/primary apoptotic cells (Annexin V positive/PI negative); upper-right represent late apoptotic/secondary apoptotic cells (Annexin V positive/PI positive); upper-left represent necrotic cells (Annexin V negative/PI positive). The numbers in the respective quadrants indicate the percentage of cells present in that area. Data shown represent one of three independent experiments.

Article Snippet: The ability of peptide B11 to induce cell death in terms of apoptosis was determined using flow cytometry with Annexin V-FITC apoptosis detection kit (Beijing Beyotime Corp, Beijing, China) following the manufacturers’ instructions.

Techniques: Inverted Microscopy, Staining, Fluorescence, Microscopy

Journal: Molecules

Article Title: A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells’ Proliferation

doi: 10.3390/molecules23123202

Figure Lengend Snippet: Localization of peptide B11 in the mitochondria, its effect on mitochondrial membrane potential (∆Ψm), and apoptosis induction in HeLa cells. ( A ) Microscopic images showing the intracellular localization of rhodamine-labeled B11 in HeLa cells. Cells were treated with 50 μg/mL of rhodamine-labeled B11 for 8 h, washed with PBS and stained with 200 nM of MitoTracker Green. Images were captured with a confocal microscope under a 40× objective (scale bar = 10 µm). ( B ) Mitochondrial membrane potential (∆Ψm) of HeLa cells treated with peptide B11. Cells were treated for 24 h with peptide B11 (50 μg/mL) and PBS (0.01 M, pH 7.4), followed by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1) working solution and incubated for 20 min at 37 °C protected from light. Images were observed under confocal microscopy (scale bar = 20 µm). For positive control, cells were treated with 10 µM of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating an intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of the ∆Ψm. ( C ) Immunoblots of ( i ) caspase-9 and caspase-3, ( ii ) Bax, and ( iii ) Bcl-2 protein levels in peptide B11-treated HeLa cells analyzed by Western blot. Cell lysates from HeLa cells treated with peptide B11 (50 μg/mL) or PBS (0.01 M, pH 7.4) for 24 h were analyzed using the appropriate antibodies, with β-actin used as a loading control. Numbers below the blots represent the relative gray values determined using ImageJ program.

Article Snippet: The ability of peptide B11 to induce cell death in terms of apoptosis was determined using flow cytometry with Annexin V-FITC apoptosis detection kit (Beijing Beyotime Corp, Beijing, China) following the manufacturers’ instructions.

Techniques: Membrane, Labeling, Staining, Microscopy, Incubation, Confocal Microscopy, Positive Control, Fluorescence, Western Blot, Control