Fig 2: The proliferation of human bladder T24 (ATCC® HTB-4™) cells subjected to S1P. An increase in the total number of cells incubated in the presence of S1P at concentrations ranging from 0.1 µM to 5 µM for 48 (grey bars), 72 (blue bars), and 96 h (brown bars), the confluence of respective cell populations and number of Ki67-positive cells when compared to untreated control are presented in panels (A–C), respectively. In panel (D) representative microphotographs of the S1P-treated cellular population upon culturing for 48, 72, and 96 h are shown. Alterations in cellular confluence are presented using bright field (upper row) and pseudocolour (lower row). Scale bar equals 10 µm. For panels (A–C) results are presented as mean ± SD from 3 repetitions. For panel (D) the results of one representative experiment are shown. * indicates statistical significance (p < 0.05) when compared to untreated control cells.

Fig 3: Restriction of human bladder T24 (ATCC® HTB-4™) cells proliferation upon FTY720P addition. The confluence of S1P-treated human bladder T24 (ATCC® HTB-4™) cell population, when preincubated with FTY720P (10 µM), as well as number of Ki67-positive cells is presented in panels (A,B). Representative microphotographs of the cellular population treated with S1P (0.1 µM) or pre-treated with FTY720P (10 µM) and then cultured in the presence of S1P (0.1 µM) for 48 (grey bars), 72 (blue bars) and 96 h (brown bars) are presented in panel C. Alterations in cellular confluence are presented using bright field (upper row) and pseudocolour (lower row). Scale bar equals 10 µm. For panels (A,B) results are presented as mean ± SD from 3 repetitions. For panel (C) the results of one representative experiment are shown. * and # indicate statistical significance (p < 0.05) when compared to untreated control cells and S1P-treated cells, respectively.

Fig 4: S1P-stimulated induction of LL-37 production in urinary bladder cells. Effect of S1P and FTY720P on LL-37 release from stimulated T24 (ATCC® HTB-4™) cells is shown in panel. Cells were incubated with indicated agents for 24 (grey bars), 48 (blue bars), and 96 h (brown bars). Results are presented as mean ± SD from 2 duplicates. * and # indicate statistical significance (p < 0.05) when compared to untreated control cells and S1P-treated cells, respectively.

Fig 5: Cytotoxicity evaluation of pleurotin to human skin fibroblasts (PB-AI001-F-DT1) and T24 bladder-carcinoma cells (ATCC HTB-4). CyQuant LDH Cytotoxicity Assay. Maximum = maximum lysis buffer (positive control). Spontaneous = 10 μL water (negative control). Optical density measured at 490 and 680 nm and % cytoxicity calculated. N = 4, error bars indicate mean ± standard error. Estimated Marginal Means with a Holm–Bonferroni adjustment was used for pairwise comparisons: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.