Xav939

HFDPCs were seeded at a density of 5×10⁵ cells per 100-mm petri dish. The cells were allowed to attach overnight and then the growth medium was replaced with a fresh one containing 0 and 10 µM of TCQA or 0.1 µM minoxidil used as positive control (Tokyo Chemical Industry, Tokyo, Japan). HFDPCs were treated as well with only 10 µM XAV939 (SIGMA, Saint Louis, USA), then with 10 µM XAV939 following by a further incubation with 10 µM TCQA (XAV939/TCQA), and finally co-treated with 10 µM XAV939 and TCQA simultaneously (XAV939+TCQA). After 6 and 12 h treatment, the growth medium was removed, and the cells washed with cold PBS before the total RNA was extracted using ISOGEN kit (Nippon Gene, Tokyo, Japan) following the manufacturer’s instructions. The RNA concentration was assessed using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Massachusetts, USA).

HPDLCs at a density of 2 × l0⁵/mL were inoculated on three 96-well plates [20 (link)] in an incubator at 37 °C with 5% CO₂ until 70~80% confluence was reached. Each group in this study had six multiple holes and the surrounding holes were filled with sterile PBS solution. Luteolin (batch number: 111520–200,201, purity> 99%, China Food and Drug Administration, Beijing, China) was dissolved by dimethyl sulfoxide (DMSO, Sigma, USA), and the different concentrations (0.01, 0.1, 1, 10, 100 μmol/L) of Luteolin [21 (link), 22 (link)], Wnt/β-catenin pathway inhibitor XAV939 (5 μmol/L, purity> 98%, Sigma, USA) [23 (link)], 1 μmol/L of Luteolin in combination with XAV939 (Luteolin was first incubated for 20 min and added with XAV939) were added into well plates and served as positive groups. In addition, untreated cells in control group were incubated with PBS buffer and considered as a negative control, compared with other groups in this study. The treated cells used in the following experiments were confirmed by preliminary experiments, as well as the time frame had the best experimental effect at the corresponding treatment time.

The DLX-6AS1 overexpressing vector (pcDNA3.1-DLX6-AS1) and the control vector (pcDNA3.1) were purchased from Genescript company (Nanjing, China). The siRNAs for DLX6-AS1 (DLX6-AS1 siRNA#1 and #2) and the control scrambled siRNA as a negative control were purchased from Ribobio company (Guangzhou, China). The chemical reagents including XAV939 (Wnt/β-catenin pathway inhibitor), lithium chloride (LiCl; Wnt/β-catenin activator) and SB-216763 (Wnt/β-catenin activator) were both purchased from Sigma, and the bladder cancer cells were pre-treated with XAV939 (10 μM), LiCl (20 mM) or SB216763 (30 µM) for 24 h before further transfection studies. For the cell transfection studies, cells were transfected with the corresponding plasmids and siRNAs using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) by following the manufacturer’s instructions. At 24 h after transfections, cells were prepared for further in vitro assays.