Journal: Scientific Reports
Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice
Figure Lengend Snippet: Generation of hepatic stellate cell-specific Cygb-transgenic mice. ( a ) DNA construct map for Cygb-mCherry. The Cygb-2A-mCherry reporter was generated with the BAC clone RP23-330N7A, and a partial genomic map of the Cygb gene with coding exons (black boxes), noncoding regions including the promoter of the Cygb gene (light grey boxes), and flanking introns (solid lines) is shown. The 2A-mCherry reporter gene, which was flanked by 110 bp of the upstream sequence of the Cygb gene stop codon and 87 bp of the downstream sequence of its stop codon, was precisely transferred to the Cygb gene. ( b ) Genotyping of Cygb-transgenic ( TG) mice in the F1 generation by Southern blots showed TG mice (lanes 9, 11, 14, and 15) bearing 10 copies of the Cygb transgenes. NC, DNA negative control; PC, DNA positive controls with 1, 3, 10, and 30 copies. ( c ) Genotyping of offspring by real-time qRT-PCR. DNA isolated from tail biopsies of Cygb 10 copies-transgenic founder (TgFD10c) mice was used as a positive control, and DNA from the 1-copy founder (TgFD1c) was used as a reference sample. Relative quantification of Cygb (green bars) and mCherry (red bars) DNA is shown. Mouse numbers BAC 687, 690–694, and 696 were clarified as Cygb-TG, and the remaining mice were WT. The relative number of DNA copies was normalized to Gapdh levels. ( d ) Macroscopic view of multiple organs of WT and Cygb-TG mice under a contrast photo (right panel) and fluorescence images of Cherry (left panel). ( e ) Real-time qRT-PCR analysis shows the Cygb expression levels in multiple organs of WT (white bars) and TG mice (green bars). Transcriptional levels of only mCherry were examined in TG mice (red bars). Gapdh was used as an endogenous control. ( f ) Immunoblot analysis showed the CYGB and mCherry protein levels in multiple organs of WT and TG mice. GAPDH was used as the loading control and for normalization. Full-length Western blots in one gel are presented in Supplementary Fig. S6 . ( g ) Representative imaging of CYGB (green) and mCherry (red) immunofluorescence staining from WT and TG livers. Br, Brain; Li, Liver; Panc, Pancreas; He, Heart; Lg, Lung; Int, Intestine; Sp, Spleen; Kid, Kidney. P, portal vein; C, central vein.
Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).
Techniques: Transgenic Assay, Mouse Assay, Construct, Generated, BAC Assay, Sequencing, Negative Control, Quantitative RT-PCR, Isolation, Positive Control, Fluorescence, Expressing, Western Blot, Imaging, Immunofluorescence, Staining