Cxcr4

The cells and tissue specimens were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor (CoWin Biosciences). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science and Technology Co., Ltd. Beijing, China). Each sample with equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After running 1–2 h under 120V, the protein was transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% not-fat milk for 1 h at room temperature and incubated with primary antibodies c-Myc, HIF-1α, CXCR4, SDF-1, vimentin, E-cadherin, and β-actin overnight at 4°C (Affinity Biosciences). Subsequently, the membranes were washed 3 times with tris-buffered saline and Tween-20 and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA, USA). Finally, the membranes were treated with a chemiluminescence reagent and exposed to X-ray. The band intensities were quantified using image J software (National Institutes of Health, Bethesda, MD, USA). Relative protein expression was quantified using control protein β-actin.

QLY granules were prepared by the Institute of Chinese Medicine, Anhui Academy of Medical Sciences. Berberine (batch No. 0713-200107), Oxymatrine (batch No. 110780-200405), Sinomenine (batch No. 0774-200206) were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Tripterygium glycosidorum (TG) tablets (batch No. 2002103B) were purchased from the Zhejiang Deende Medicine Co., Ltd. (Zhejiang, China). Bacillus Calmette-Guerin (BCG) was obtained from REBIO (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β, tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-2 and IL-6 were bought from Elisa Biotech (Shanghai, China). Rabbit anti-mouse β-actin, CXCL12, CXCR4, P65, p-P65 were purchased from Affinity Biosciences (Cincinnati, OH, USA).