Fig. 1. CDDP and 5-FU induce CEBPD and contribute to myofibroblast differentiation.

A, B MTT assays were conducted to assess the cytotoxic effect of CDDP or 5-FU on HFL1, 7V7, and KO5 cells. HFL1 cells were infected with shβ-galactosidase (shC) or shCEBPD (shD) lentiviruses. Cells were seeded and their growth activity after treatment with CDDP or 5-FU for 24 h was compared. C An RT-PCR assay was performed with total RNA harvested from HFL1 cells treated with 30-μM CDDP, 10-μg/ml 5-FU, or 5-ng/ml TGFβ1. D HFL1 cells were infected with shC or shD lentiviruses and treated with or without CDDP or 5-FU for 6 h. For gain-of-function assays, total RNA was harvested from HFL1 cells infected with lentivirus bearing pAS3W-control (Ctl) or pAS3W-CEBPD expression vectors (CD). CEBPD, α-SMA, TGFβ1, and GAPDH transcripts were examined via RT-PCR. E HFL1 cells were infected with shC or shD lentiviruses and treated with or without CDDP or 5-FU for 6 h. F HFL1 cells were infected with lentivirus bearing Ctl or CD expression vectors. α-SMA expression and the formation of actin stress fibers were detected via immunofluorescence microscopy. G A549 cells mixed with HFL1 cells carrying a shβ-galactosidase knockdown vector with GFP (shC-GFP) or a CEBPD knockdown vector with GFP (shD-GFP) 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 α-SMA (red) and GFP (green), and nuclei were stained with DAPI (blue). Three independent experiments were performed in triplicate. All 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.