Fig. 4

Lactate-induced LCN2 expression enhances bladder cancer cells migration. (a) 5637 cells were cultured in the absence and presence of 10 mM lactic acid for 24 h. Cells were then collected and subjected to trans-well assay to evaluate cell migration ability. Representative images of migrated 5637 cells, stained with crystal violet, were captured by an inverted microscope (100x magnification; Scale bar: 200 μm) are shown here. The accompanying histogram quantifies the number of migrated cells per field, showing a significant increase in migration following lactic acid treatment (p < 0.01). (b) Cell lysates from (a) were collected to examine vimentin, N-cadherin and E-cadherin expressions. b-actin was used as a loading control. (c) Examination of vimentin, N-cadherin and E-cadherin expression in scrambled shRNA control or LCN2-silenced 5637 cells (LCN2 KD). (d) Scrambled shRNA control and LCN2-silenced 5637 cells were subjected to a trans-well assay to examine the role of LCN2 in regulating cell migration. Similar experiments were performed as shown in (a). Migrated cells were stained with crystal violet and subsequently quantified (p < 0.01). (e) A scratch assay was performed to evaluate the wound healing ability of control, lactic acid-treated control, and LCN2-silenced 5637 cells (LA + LCN2 KD) over a 9 h incubation period. Cells were pre-treated with 10 mM lactic acid for 24 h, while untreated cells served as the control. Cells migration was photographed at 0, 3, 6, 9 h using an inverted microscope at a 40x magnification (Scale bar: 100 μm). The percentage of wound closure was quantified relative to 0 h time point. Results expressed as mean ± SEM (*p < 0.05, **p < 0.01).