Ham s f 12 kaighn s modification medium

THP-1 cells were cultured in RPMI 1640 medium (Hyclone). 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). The CAF/F28 was established from a patient [45 (link)]. Mouse embryo fibroblasts (MEFs) used in this study were isolated from individual E13.5-E14.5 embryos generated by mating Cebpd null heterozygous mice and immortalized with E1A [46 (link)]. The CAF/F28 cells, human breast cancer MB231 cells, mouse breast cancer 4T1 cells and immortalized Cebpd^+/+ (7V7) and Cebpd^−/− (KO5) MEFs were maintained in DMEM. All culture media were supplemented with 10% FBS, streptomycin and penicillin. To generate THP-1/M2 macrophages, THP-1 cells were treated with 320 nM PMA for 6 h and then cultured with PMA plus 20 ng/ml IL-4 and 20 ng/ml IL-13 for another 18 h [47 (link)]. To generate primary mouse M2 macrophages, mononuclear cells obtained from bone marrow were treated with 25 ng/ml M-CSF in RPMI 1640 medium with 10% FBS [48 (link)]. To generate myofibroblasts, HFL1 cells or MEFs were treated with 5 μg/ml TGF-β for 3 days. The following concentrations of peptide were used in this study: 10 μg/ml PTX3 peptide (RI37 or KT44). The anticancer drugs were then added individually for each experiment: 40 μM CDDP or 100 μM 5-FU.

All cell lines were obtained from and authenticated by the American Type Culture Collection (ATCC). ATCC authentication of cell lines includes assessment of cell morphology, karyotyping, and short tandem repeat profiling. Cells were cultured under standard conditions (37 °C and 5% CO₂) in sterile, TC-treated, nonpyrogenic, polystyrene tissue culture dishes (Cat#: Corning 430167). HEK-293 (Cat#: ATCC CRL-1573), HEK-293T (Cat#: ATCC CRL-3216), and HeLa (Cat#: ATCC CCL-2) cells were grown in DMEM (Corning, Cat#: 10-013-CV) supplemented with 10% FBS (VWR, Cat#: 89510-186) and 1% Pen/Strep (Gibco, Cat#: 15140-122). A549 cells (Cat#: ATCC CCL-185) were maintained in Ham’s F-12 Kaighn’s Modification medium (Gibco, Cat#: 21127-022) supplemented with 10% FBS and 1% Pen/Strep. All cell lines tested negative for mycoplasma using the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich). Mycoplasma testing was carried out on 1 mL of cell culture media taken from tissue culture dishes containing confluent monolayers of cells on a routine basis at least twice a year.

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.