Fig. 4

TNF inhibits survival and muscle functional gene expression of lymphatic muscle cells. a LMCs were isolated from rat mesenteries. Expression of genes specific for smooth muscle cells, α smooth muscle actin and smooth muscle myosin heavy chain 2 (sMYH2), was determined by immunostaining and Western blot analysis, respectively. C2C12 myoblast precursor cells and LECs were included as negative controls. b LMCs were treated with 10 ng/ml TNF. Cell growth was assessed by an MTT assay. Values are mean ± SD of 3 samples. *p < 0.05 by analysis of variance in repeated measurement design followed the LSD-t post hoc test. c Cell apoptosis was determined by flow cytometry. d LMCs were treated with TNF for 24 h. Expression levels of muscle functional genes were determined by qPCR. The fold changes were calculated using PBS-treated cells as 1. Values are mean ± SD of 3 samples. *p < 0.05 by the Student t test. e LMCs were treated with 20 ng/ml TNF and 20 ng/ml IL-6 for 24 h. The protein levels of sMYH2 and h1-Calponin were determined by Western blot analysis. f LMCs were treated with 10 ng/ml TNF for various time points. The phosphorylation of ERK1/2 was examined by flow cytometry. Experiments were repeated 3 times with similar results