PN promotes cell proliferation of CCA cell lines. KKU-M156, KKU-M213 and KKU-M055 showed significantly induced proliferation with different concentrations of PN (A-C) whereas KKU-100 was unresponsive to PN (D). With an optimal dose of PN for each cell line, KKU-M213, KKU-M156, and KKU-M055 increased cell proliferation in a time-dependent manner (E). Triplicate experiments were performed for each assay. Results are expressed as mean ± SD and an asterix represents a P value less than 0.05 when compared to the negative controls without PN treatment. Black and dashed lines represent cells with and without PN treatment, respectively. Colony formation assay with and without agar was performed (F). Colony of more than 30 cells was counted under inverted microscope. Numbers of colonies/well in 6-well plate of both KKU-M213 and KKU-M156 CCA cell lines were higher in condition of PN treatment than those without PN. Results are expressed as mean ± SD of duplicate experiments. Pictures of crystal violet-stained cells are of KKU-M213 CCA cells in comparison between with and without PN.