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. 2015 Jan 5;235(5):710–720. doi: 10.1002/path.4494

Figure 4.

PATH-4494-FIG-0004-c

MCP‐1 induces LXRα‐dependent lipid accumulation in primary hepatocytes. (A) Hepatocytes were treated with 50 ng rhMCP‐1 protein and/or 1 µm 22‐S‐HC for 48 h. At the end of treatment, lipid droplets were stained using Nile red and visualized by fluorescence microscopy (×400). (B) Huh‐7 cells were treated with recombinant human MCP‐1 and/or 22‐S‐HC for 48 h and then their neutral lipid content was analysed by flow cytometry after Nile red staining; geometric mean fluorescence intensity values for peaks were indicated (top). (C) Huh‐7 cells were transfected with siGFP, siLXRα or siHIF‐1α and then treated with rhMCP‐1 for 48 h; their neutral lipid content was analysed by flow cytometry after Nile red staining (left); hepatocytes were infected by lenti‐shGFP or lenti‐shLXRα viruses for 24 h and then treated with 50 ng rhMCP‐1 protein for an additional 24 h; whole‐cell lysates were analysed for protein expression by western blotting (right). (D) Hepatocytes were treated with 50 ng rhMCP‐1 protein for 24 h; total RNA was prepared and analysed for expression of the indicated transcripts by qRT–PCR; numbers represent mean ± SD (n = 3); *p < 0.05 and **p < 0.01 compared with vehicle‐treated control; # p < 0.05 compared with the MCP‐1 treatment with shGFP transfection. (E) Huh‐7 cells were transiently transfected with the indicated human LXRα promoter–Luc (left and centre) or LXRE‐tk‐Luc (right); transfected cells were treated with rhMCP‐1 for 24 h; numbers represent mean ± SD (n = 3); **p < 0.01 and ***p < 0.01 compared with the vehicle‐treated control; statistical significance was evaluated by two‐way ANOVA. (F) Huh‐7 cells were transfected with siLXRα or siHIF‐1α for 24 h and then treated with rhMCP‐1 for an additional 24 h. DNA fragments that immunoprecipitated using the indicated specific antibodies were amplified by PCR, using specific primers for LXRα promoter (top) or the SREBP‐1 promoter (bottom); numbers represent mean ± SD (n = 3); *p < 0.05 and **p < 0.01 compared with vehicle‐treated control; statistical significance was evaluated by two‐way ANOVA