Fig. 6

PNS blocks the inhibitory effects of TNF on lymphatic smooth muscle cells via lymphatic endothelial cells. a LMCs were treated with PBS or 10 ng/ml TNF ± 100 ng/ml PNS for 24 h. The expression of muscle functional genes was examined by qPCR. The fold changes were calculated using PBS-treated cells as 1. Values are mean ± SD of 3 samples. b LECs were treated with PBS or TNF ± PNS for 24 h. NO levels in the conditioned medium were assessed by the Greiss method. Values are mean ± SD of 3 samples. c LECs were pre-treated with PBS or TNF ± PNS for 24 h and were then co-cultured with LMCs for an additional 24 h. The expression of muscle functional genes in LMCs was determined as in a. d LMCs were treated with PBS or 10 ng/ml TNF ± 100 ng/ml PNS for 24 h. Annexin V+/propidium iodide+ apoptotic cells (%) were examined by flow cytometry, as in Fig. 4. e LECs were pre-treated with PBS or TNF ± PNS or 1 mM Ami for 24 h and were then co-cultured with LMCs. Apoptotic cells (%) were determined as in b. Values are mean ± SD of 3 samples. Three experimental repeats. *p < 0.05 vs. PBS-treated, ^#p < 0.05 vs. PNS-treated cells by one-way analysis of variance followed by the Dunnet post hoc test. f A working model showing how PNS affects LMCs in the presence of TNF