Cell culture. Human adult dermal fibroblasts (PromoCell GmbH, Heidelberg, Germany), HeLa cells (ATCC, Manassas, VA, USA) and human umbilical-cord–derived mesenchymal stem cells (HUC-MSCs) (PromoCell GmbH, Heidelberg, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 0.1% antibiotics (Gibco, Grand Island, NY, USA) at 37 ºC in a 5% CO2 incubator (APM-30D; ASTEC, Fukuoka, Japan)10. Human umbilical vein endothelial cells (HUVECs) (Lonza, Warkerville, MD, USA) were cultured in endothelial cell growth medium-2 (PromoCell GmbH, Heidelberg, Germany). An oxygen level of 21% as a normoxic condition and 1% O2 as a hypoxic condition were applied to the culture condition of HDFs from passage 4 to passage 610. When cell confluency of all cells reached 90%, the cells were passaged using 0.25% Trypsin-EDTA (Gibco, Grand Island, NY, USA). For the proliferation assay of both culture conditions of HDFs, 2 × 105 HDFs at passage 6 were cultured in a 100-mm culture plate for 5 days, and cell numbers were measured using Trypan blue 0.5% solution (Biowest, Riverside, MO, USA) staining10.
Preparation of CM from normoxic and hypoxic HDFs. Normoxic and hypoxic HDFs (2 × 105 cells) at passage 6 were cultured in 100-mm culture plates with complete medium. When cell confluency reached 90% at day 5, the cultured medium was removed and 1× phosphate-buffered saline (PBS) was added to wash the cells. Next, 6 mL of DMEM without FBS or antibiotics was added to the cells. After incubation for 24 h, N-CMs and H-CMs were harvested and centrifuged at 300 g for 5 minutes. The supernatant was transferred to new 15 mL tubes and stored at − 80 ºC. For a control, CM (C-CM), DMEM without FBS or antibiotics was used10.
Cell viability assay. Normoxic and hypoxic HDFs (1 × 103 cells) at passage 6 were seeded in 96-well white plates. After 5 days of culture, 100 µL of CellTiter-Glo assay 2.0 reagents (Promega, Madison, WI, USA) was applied to the cells. After 10 min of incubation, the luminescence ratio indicating cell viability was measured using a GLOMAX Multi Detection System (Promega Biosystems Sunnyvale, CA, USA)10. For analysis of HeLa cell viability and HUC-MSCs and HUVECs treated with CM from HDFs, 1 × 104 cells were seeded in 96-well white plates. The cultured medium was removed the following day and CMs were applied to the cells. After 48 or 72 h, the same procedure using CellTiter-Glo assay 2.0 reagents (Promega, Madison, WI, USA) was followed10.
Apoptosis assay. HeLa cells (1.5 × 105) were seeded in 6-well culture plates10. The next day, the culture medium was removed and cells were treated with 2 mL of C-CM, N-CM and H-CM. After 48 h of incubation, the cells were harvested with 0.25% Trypsin-EDTA (Gibco, Grand Island, NY, USA) and stained with a fluorescein isothiocyanate annexin-V apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Stained cells were analyzed with a caliber flow cytometer (Becton-Dickinson, San Jose, CA, USA) and Flowjo software (Treestar, San Carlos, CA, USA)10.
Caspase 3/7 activity assay. HeLa cells (1 × 104) cells were seeded in 96-well white plates10. The next day, the culture medium was removed and 100 µL of C-CM, N-CM and H-CM was applied to the cells. After 12, 24, and 48 h of incubation, 100 µL of Caspase-Glo 3/7 Assay reagent (Promega, Madison, WI, USA) was added to the cells, which were then incubated for 1 h. The luminescence ratio indicating caspase 3/7 activity was analyzed using a GLOMAX Multi Detection System (Promega Biosystems Sunnyvale, CA, USA)10.
Mitochondrial membrane potential assay. HeLa cells (1 × 104) were seeded in 96-well clear plates10. The culture medium was removed the next day and 100 µL of C-CM, N-CM, and H-CM was applied to the cells. After 12, 24, and 48 h of incubation, the cells were stained with an Orange Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK). The fluorescence ratio (Ex/Em = 540/590 nm) indicating matrix metalloproteinases (MMPs) was measured using a Mithras2 LB 943 Multimode Reader (Berthold Biotechnologies, Bad Wildad, Germany)10.
Cell-cycle assay. HeLa cells (1.5 × 105) were seeded in 6-well culture plates10. After overnight incubation, the culture medium was removed and cells were treated with 2 mL of C-CM, N-CM, and H-CM. After 24 and 48 h of incubation, cells were harvested with 0.25% Trypsin-EDTA and fixed with 70% alcohol at 4°C for 1 h. Fixed cells were stained with 20 µg/mL propidium iodide (PI; Abcam) and 1% RNase A (QIAGEN, Valencia, CA, USA) for 30 min at 37°C. Stained cells were suspended in PBS and analyzed using a FACSVerse flow cytometer (BD Biosciences) and Flowjo software (Treestar, San Carlos, CA, USA)10.
Analysis of secretory protein by protein antibody array. Secretory proteins in N-CM and H-CM were analyzed using a RayBio Label-based (L-Series) Human L1000 Antibody Array (Raybiotech, Inc., Norcross, GA, USA) by E-biogen (Kyung Hee Business Center, Kyung Hee University, Seoul, Korea), and data were analyzed in Genowiz 4.0 (Ocimum Biosolutions, India)10. Up- and down-regulated proteins in H-CM compared with N-CM (> 2-fold) were described using UniProt DB, and GO and KEGG pathway of proteins were determined using Database for Annotation, Visualization and Integrated Discovery (DAVID) (p < 0.01)10.
Analysis of intracellular signaling pathways by protein antibody array. HeLa cells (2 × 105) cells were cultured in 100-mm culture plates with complete medium. When cell confluency reached 90%, the culture medium was removed and 1× PBS was added to wash the cells. After removing the PBS, 10 mL of N-CM and H-CM was applied to the cells for 24 h. The cells were then harvested and intracellular proteins analyzed with a Signaling Explorer antibody array (Full Moon BioSystems, Sunnyvale, CA, USA) by E-biogen (Kyung Hee Business Center, Kyung Hee University, Seoul, Korea)10. The data were analyzed using Genowiz 4.0), and up- and down-regulated proteins were described using UniProt DB. GO and the KEGG pathways of up- and down-regulated proteins were analyzed using DAVID (p < 0.01)10. Protein-protein interaction (PPI) was analyzed using the STRING database (string-db.org) and Cytoscape software (www.cytoscape.org). The number of nodes (protein) and edge (protein interaction line) were analyzed, and nodes with more than 5 edges in PPI were defined as hub proteins.
Statistical analysis. All experimental data were analyzed by the t test. A p value < 0.05 was considered statistically significant. All analyses were carried out using GraphPad Prism version 6.01 (San Diego, CA, USA).