Fig 1: Genome-wide analysis and in vivo screening identified Bst1 as a gene related with kidney fibrosis. (A) Characteristics of primary renal fibroblasts. Fibroblasts isolated from kidneys of untreated, unoperated mice, were cultured and passages 4–10 were used for all experiments. Immunocytochemistry was performed to confirm that the isolated cells were fibroblasts. An elongated, spindle-shaped morphology (phase contrast, top panels) and strong vimentin staining (red immunofluorescence, bottom panels) were observed. Anti-Vimentin Ab + : anti-vimentin, 1:250, rabbit monoclonal, ab92547, Abcam, followed by donkey anti-rabbit-Cy3, 1:500, Jackson ImmunoResearch; Anti-Vimentin Ab-: primary antibody omitted. Scale bar = 400 µm. (B) Venn diagram based on RNA-seq and ChIP-seq of primary renal fibroblasts isolated from kidneys of untreated, unoperated WT, Sphk1–/– and Sphk2–/– mice. From 203 genes identified by RNA-seq that have suppressed expression in Sphk2–/– cells (the overlap of comparison of Sphk2–/– cells to WT and Sphk1–/–cells, left), 30 genes that have lower histone acetylation at two sites (lysine 27 and lysine 9) on histone 3 (H3K27 and H3K9, respectively) in Sphk2–/– cells were chosen by ChIP-seq (the overlap of comparison of Sphk1–/– and Sphk2–/– cells, right). (C) In vivo screening. Heat map based on expression analysis of common fibrosis-related genes and the top 50% of selected genes in panel (A) (RT-PCR of RNA isolated from kidneys after unilateral IRI). Clustering was performed based on relative gene expression. Relative expression was calculated as the ratio of gene expression in the left kidney (IRI side) compared to the right kidney (non-IRI side) of wild-type mice. n = 5–7 in panel (C). Gene abbreviations defined in Supplementary Table 3 and Supplementary Figure 3.