Intercellular communication between hepatic stellate cells and myofibroblasts mediated by osteopontin and FGF18 promotes liver fibrosis. Seki et al
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Seki et al Figure 1. 1D, 1E, 1F, RNA-seq data Seki et al Figure 2. 2B, counted cell numbers 2C, 2D, qPCR 2E, 2F, ELISA 2G, qPCR Seki et al Figure 3. 3A, 3B, 3C, 3D, 3E, qPCR Seki et al Figure 4. 4B, 4D, 4F, 4H, counted cell numbers Seki et al Figure 6. 6A, 6B, 6C, 6D, qPCR Seki et al Figure S1. S1A, S1B, S1C, RNA-seq data
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1. Primary Hepatic Stellate Cells (HSCs) Primary HSCs were isolated from wild-type female mice (>24 weeks old) using a standard Nycodenz density gradient centrifugation protocol. Cells were stimulated with the indicated agents under specified conditions. Gene expression was assessed by quantitative PCR (qPCR) as shown in Figures 2C, 2D, 2G, 3A, 3B, 3C, 6C, and 6D. Osteopontin protein levels were measured by ELISA (Figures 2E and 2F). Immortalized HSCs (SV40 T antigen-transformed) were used only in Figures 3D and 3E. Primary HSCs were cultured without treatment for the indicated time periods. The expression of desmin and α-SMA was evaluated by immunohistochemistry, and the number of positive cells was manually counted (Figure 2B). 2. Liver Tissue Imaging Liver tissue sections from mice of the indicated genotypes were immunostained with the indicated antibodies. Colocalization analysis was performed using the BZ-X700 All-in-One fluorescence microscope (KEYENCE), and total cell counts were obtained using the hybrid cell counting function of the BZ-X700 system (Figure 4B, 4D, 4F, and 4H). 3. Primary Hepatocytes Primary hepatocytes were isolated using a modified two-step collagenase perfusion method. Cells were treated with the indicated agents under defined conditions. Gene expression was evaluated by qPCR (Figures 6A and 6B). 4. RNA-Seq Data Analysis Publicly available bulk RNA-seq datasets were retrieved from the indicated repositories. Correlation analyses were performed using Pearson’s correlation coefficient (Figures 1D, 1E, 1F, S1A, S1B, and S1C).
Institutions
- Toho DaigakuTokyo, Ota-ku