Fig. 3
Impaired STAT3 function attenuates lactate-induced LCN2 expression. (a) and (b) Nuclear expression of STAT3 under lactic acid treatment by immunofluorescence analysis. Cells were treated with 10 mM lactic acid for 24 h. Following treatment, cells were washed with PBS, fixed, permeabilized, and stained with anti-STAT3 antibody, followed by iFluor 594 secondary antibody. The nuclear stain is DAPI (blue). Quantification of nuclear STAT3 in the absence (control) and presence of lactic acid treatment in 5637 and MC-T2 cells was performed. Scale bar: 20 μm. *p < 0.05, **p < 0.01 (c) 5637 and MC-T2 bladder cancer cells were stably transduced with either scrambled shRNA control lentivirus or shRNA lentivirus against STAT3 (STAT3 KD). STAT3 and LCN2 expression levels were confirmed by western blot analysis. (d) Examining the induction efficiency of lactic acid treatment on LCN2 expression in STAT3-silenced 5637 cells. Scrambled control and STAT3 knockdown (STAT3 KD) cells were treated with 10 mM lactic acid for 24 h. Cell lysates were harvested for western blot analysis. (e) Inhibition of STAT3 activity by BBI608 inhibitor suppresses lactic acid-induced LCN2 expression. Cell lysates of control and 10 mM lactic acid-treated 5637 and MC-T2 cells were treated with or without 1µM STAT3 inhibitor (BBI608) for 24 h. Western blot analysis was performed to assess STAT3 activity (p-Y705) and LCN2 expression.
