Fig. 2: Activation of CEBPD in fibroblasts contributes to angiogenesis and metastasis of lung cancer.

A HUVEC proliferation and migration were assessed using MTT and Boyden chamber assays, respectively. HUVECs were cultured in conditioned medium from HFL1 cells infected with lentivirus bearing pAS3W-control (Ctl) or pAS3W-CEBPD expression vectors (CD). The angiogenic effect was assessed by counting the number of intersections between branches of HUVECs grown in conditioned medium. The data are expressed as the mean ± S.D. Statistical analysis was performed using the Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group. B Assays to assess proliferation, migration, and in vitro tube formation were conducted. HUVECs were cultured with conditioned medium from HFL1 cells infected with shβ-galactosidase (shC) or shCEBPD (shD) lentiviruses as described in materials and methods. The data are expressed as the mean ± S.D. Statistical analysis was performed using one-way ANOVA followed by the Tukey’s multiple comparison test. **p < 0.01, ***p < 0.001. C Cebpd^+/+ (WT) and Cebpd^–/– (KO) mice were subcutaneously inoculated with 800-ng/ml bFGF or 150-ng/ml VEGF Matrigel plugs. After 5 days, the mice were sacrificed, and the Matrigel plugs were removed to assess the newly formed blood vessels; hemoglobin levels were measured in the plugs using a Drakin’s reagent kit (n = 3 per group). The data are expressed as the mean ± S.D. Statistical analysis was performed using the Student’s t test: ***p < 0.001 versus the control group. D A549 cells mixed with shC-GFP or shD-GFP HFL1 cells were inoculated subcutaneously into the dorsal rear flanks of NOD-SCID mice, and the mice were treated with or without CDDP (5 mg/kg). The mice with A549-xenografted tumors were sacrificed in the 12th week. Tumor tissues were stained for CD31 (red) and GFP (green), and nuclei were stained with DAPI (blue). E Tumor volume was measured with external calipers and calculated using the standard formula: V = (w × l²) × 0.52, where l is the length and w is the width of the tumor (n = 6 per group). F The number of metastasis nodules from A549-xenografted tumors in the lungs was examined. Three independent experiments were performed in triplicate. The data are expressed as the mean ± S.D. Differences among groups were analyzed with one-way ANOVA followed by the Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.