Hmec 1

Human dermal fibroblast cell line (BJ), human dermal microvascular endothelial cell line (HMEC-1) [31 (link)], and human acute monocytic leukemia cell line (THP-1) were purchased from ATCC (Manassas, VA, USA). BJ cells were cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Corning Incorporated, Corning, NY, USA) at 37 °C in a humidified 5% CO₂ atmosphere. After adhering the cells, they were starved for 24 h with 0.1% FBS in DMEM, then stimulated with human TGF-β1 (PeproTech, Rocky Hill, NJ, USA) for 24 h. HMEC-1 cells were cultured in MCDB131 (without L-glutamine) as base medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA),100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS in 70 cm² flasks until confluence at 37 °C, 5% CO₂ atmosphere. After the cells were attached, TNF-α is stimulated for 24 h. THP-1 cells were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO₂ atmosphere.

The cytotoxicity of BR2GK and its derivatives were determined by hemolysis assay and MTT assay on the dermal microvascular endothelium cell line, HMEC-1 (ATCC CRL-3243). Horse erythrocytes (TCS Biosciences Ltd., Buckingham, UK) were subjected to the hemolysis of all synthetic peptides as described previously [35 (link)]. Briefly, the erythrocytes were washed and resuspended by PBS to achieve a 4% suspension. The test peptide solutions were transferred into the aforementioned suspension and kept at a constant 37 °C for 2 h, while Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) was employed to replace the peptide solution for establishing the positive control. The supernatants after incubation were measured at 470 nm.
MTT assays were conducted as outlined previously [35 (link)]. Ten thousand cells were seeded in each well of a 96-well plate. Cells were treated with different peptide concentrations for 24 h. Triton (0.1%) X-100 was used as a positive control.

Four cancer cell lines, including Human non-small cell lung cancer cell line H157 (ATCC-CRL-5802), Human breast adenocarcinoma cell line MCF-7 (ATCC-HTB-22), Human prostate carcinoma cell line PC-3 (ATCC-CRL-1435), Human neuronal glioblastoma cell line U251MG (ECACC General Cell Collection: 09063001), and one normal cell line, Human microvascular epithelial cell line HMEC-1 (ATCC-CRL-3243), were utilized to estimate the antiproliferation effects and the cytotoxicity of Dermaseptin-PP. All cancer cells were cultured with full-growth medium supplemented with 1% penicillin-streptomycin solution (Sigma, UK) and 10% fetal bovine serum (FBS) (Sigma, UK). H157 and PC-3 cells were cultured in RPMI-1640 medium (Invitrogen, Paisley, UK), MCF-7 and U251MG cells were grown in Dulbecco's Modified Eagle's medium (DMEM) (Sigma, St. Louis, MO, USA), while HMEC-1 cells were cultured in full-growth MCDB-131 medium (Gibco, Paisley, UK) with 10 ng/mL epidermal growth factor (EGF) and 10 mM L-Glutamine.

The murine gliosarcoma cell line (9 L/lacZ), the murine endothelioma cell line (bEnd.3), and the human microvascular endothelial cell line (HMEC-1) were purchased from ATCC and cultured in DMEM medium, supplemented with 10% (FBS, Seradigm, USA) and 100 U/mL penicillin/streptomycin (Gibco, USA), at 37 °C in a humidified incubator. Cell cytotoxicity assessment methodology is described in supporting information.

Human colon epithelial cells (FHC), colon adenocarcinoma cell line (DLD-1), rectal adenocarcinoma cell line (HT29), colon cancer cell line (HCT116), and human microvascular endothelial cell line (HMEC-1) were purchased from ATCC, while the cecum adenocarcinoma cell line (NCI-H508) was purchased from the Center for basic Medical Cell, Institute of basic Medicine, Chinese Academy of Medical Sciences. The exosome-free fetal bovine serum (FBS) was produced by centrifugation (100,000 g) at 4°C overnight in order to ensure the removal of any bovine-derived exosomes [35 (link)]. The FHC cells were cultured in DMEM/F12 (Hyclone) medium containing 10% FBS. The DLD-1 cells were cultured in RPMI1640 medium containing 10% FBS, HT29 while the HCT116 cells were cultured in McCoy's 5A medium containing 10% FBS. The NCI-H508 cells were cultured in DMEM medium containing 10% FBS, and HMEC-1 cells were cultured in DMEM medium (31600-034, Hyclone, USA) comprised of 10% FBS (10099141, Gibco, USA) as well as streptomycin mixture. All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. When cell confluency reached 90%, the cell passage procedure was performed accordingly.

The human neuronal glioblastoma cell lines, U251MG (ECACC-09063001), the human breast cancer cell lines MCF-7 (ATCC-HTB-22), the human pancreatic cancer cell lines, PANC-1, the non-small cell lung cancer cell line NCl-H157 (ATCC-CRL-5802) and the human prostate carcinoma cell line PC-3 (ATCC-CRL-1435) were selected to test the cytotoxicity of the peptides. The human microvascular endothelial cell line, HMEC-1 (ATCC-CRL-3243) was utilized to evaluate the cytotoxicity of the peptides on normal human cells. The cell lines were cultured as described previously [12 (link)].
The cell proliferation inhibitory rate was assessed by MTT assay as previously performed [12 (link)] with minor modifications. The peptide was diluted to a final concentration gradient from 1 µM to 100 µM, treating the cell lines for 24 h.