Figure 4
KLF4 gene expression was tightly linked with that of FSP-1 in fibrocyte generation from MDSCs. (a) Splenocytes from Rosa26CreER/KLF4 (flox+/+) mice were purified and subjected to fibrocyte generation using a recently developed approach with the application of murine interleukin (IL)-13 (50 ng/ml) and macrophage colony-stimulating factor (25 ng/ml).23 4-OH tamoxifen (4-OH TAM, Sigma-Aldrich) was used to induce KLF4 knockout in vitro and 100% ethanol as a control. Three days later, the cells were stained with Hema 3 staining kit (Fisher Scientific, Waltham, MA, USA) and the numbers of fibrocytes per 1 × 105 splenocytes in each group were counted. Left, representative photographs of fibrocytes generated from splenocytes (red arrows) were shown. Right, the number of fibrocytes per 1 × 105 splenocytes in each group was counted and compared (n=5). (b) The total RNA of fibrocytes generated as described in a were extracted using Trizol Reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis and quantitative reverse transcriptase–PCR (qRT–PCR) were performed as described previously.15 The relative expression levels of KLF4 and FSP-1 were normalized to that of GAPDH. Primer sequences for qRT–PCR were listed in Supplementary Table S1. (c) Four different MDSC subsets in mouse splenocytes were gated using CD11b and Ly6G antibodies (left) and then sorted for qRT–PCR (middle) and fibrocyte generation assays (right). (d) Left, KLF4 bound to the promoter region of FSP-1 by the chromatin immunoprecipitation assay as we previously described28 using NIH 3T3 cells (ATCC, Manassas, VA, USA). Two KLF4 antibodies (KLF4-1 antibody from our laboratory29 and KLF4-2 antibody from Genespin (Milano, Italy)) were used to precipitate DNA–protein complexes followed by PCR to amplify a 135-bp fragment in the FSP-1 promoter. Normal rabbit immunoglobulin G (IgG, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a negative control. The PCR primers were listed in Supplementary Table 1. Right, KLF4 overexpression increased the promoter activities of FSP-1 by transient transfection and dual luciferase assay. A ~2.3 kb mouse FSP-1 promoter region was amplified (MGI: 1330282, nucleotide position −1104 to 1224) using the primers listed in Supplementary Table 1. It was cloned into pGL2 basic vector (Promega, Sunnyvale, CA, USA) and confirmed by sequencing. FSP-1 promoter luciferase reporter and KLF4-overexpressing construct were co-transfected into NIH 3T3 cells using TurboFect (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The empty pGL2 vector was used as a control. Two days after transfection, the cells were lysed and the effect of KLF4 overexpression on FSP-1 promoter activity was assessed using the dual luciferase assay system (Promega). Mean±s.e.m., *P< 0.05, **P< 0.01.
