Hfl 1

Human fetal lung fibroblasts (HFL1; catalogue number CCL-153) and A549 (catalogue number CCL-185) were purchased from the American Type Culture Collection (Manassas, VA, USA). Lung fibroblasts and A549 cells were cultured in DMEM supplemented with 10% FCS, 100 μg/ml penicillin, 250 μg/ml streptomycin, and 1 μg/ml amphotericin B in a humidified atmosphere of 5% CO2. Sub-confluent cells were removed from the dishes by 0.05% trypsin-EDTA (Wako, Osaka, Japan). For three-dimensional collagen gel contraction and ELISA, primary lung fibroblasts were used at the fourth to sixth passages after isolation to exclude the effect of differences in passage number and culturing conditions.

Human lung fibroblasts (HFL1) were obtained from the American Type Culture Collection (ATCC). HFL1 cells were cultured in Ham’s F-12 Kaighn’s modification medium (Gibco). MEFs were isolated from individual E13.5-E14.5 embryos generated by mating Cebpd null heterozygous mice. The MEFs used in this study were immortalized by E1A^{54 (link)}. The mouse breast cancer 4T1 cells and immortalized Cebpd^+/+ (7V7) and Cebpd^–/– (KO5) MEFs were maintained in Dulbecco’s modified Eagle’s medium. All culture media were supplemented with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml), and penicillin (100 U/ml). HUVECs were purchased from the Bioresource Collection and Research Center of Taiwan and maintained in ECM (ScienCell) supplemented with 5% FBS, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin. The recombinant protein, reagents, or inhibitors were then added individually for each experiment: 0.5-μg/ml SDF4 (Abnova), 30-μM CDDP (Sigma), or 10-μg/ml 5-FU (Sigma), 100-nM wortmannin, 10-μM PD98059, 10-μM SB203580, and 10-μg/ml AMD3100.

Human fetal lung fibroblasts (HFL‐1) were purchased from the American Type Culture Collection (CCL‐153, Manassas, VA). The human lung parenchymal fibroblasts were cultured from the resected lung tissues of patients with BHDS (BHDS lung fibroblasts) and patients with lung cancer (control lung fibroblasts), using the method by Holz et al. (2004). In patients with lung cancer, only lungs without visible or palpable lung metastases were used to avoid isolating cancer‐associated fibroblasts. Briefly, the portion of lung parenchymal tissue that was free of the pleural surface was minced and placed in culture with Dulbecco's modified eagle's medium (DMEM) supplemented with 10% FCS, 100 μg/mL penicillin, and 250 μg/mL streptomycin (complete medium) in a humidified atmosphere of 5% CO₂ and passaged every 4–5 days at 1:4 ratios. They were used for chemotaxis and three‐dimensional (3‐D) collagen gel contraction assays at the fourth to fifth passages after isolation to exclude the effect of differences in passage number.

Human ovarian cancer cell lines (TOV112D, TOV-21G, SKOV3, MDAH2774), lung fibroblast cells (HFL1), and normal aorta smooth muscle cells (T/G HA-VSMC) were obtained from the American Type Culture Collection (Manassas, VA, USA). TOV112D, TOV-21G, SKOV3, MDAH2774, and HFL1 were cultured in DMEM/F12 with 10% fetal bovine serum and appropriate amounts of penicillin and streptomycin at 37°C with 5% CO2. T/G HA-VSMC cells were cultured in DMEM/F12 with 10% fetal bovine serum, 0.05% mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite, and 0.03 mg/ml endothelial cell growth supplement. For apoptosis analysis, cells transfected with BAIAP2L1 siRNA were either irradiated with UV (100 J/M²) or treated with 10 μM cisplatin (Fresenius Kabi, NC, USA) for 24 hrs before analysis.