Drug and reagents
NDQ [National drug standard No: WS3-229(Z-033)-2000(Z)] was obtained from Guangzhou Kangchen Pharmaceutical Co., Ltd. (Guangzhou, China), and granules were dissolved in PBS at a concentration of 1.25 g/ml.
TGF-β1 peptides was purchased from Biosynthesis biotechnology (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) was obtained from Thermo Fisher Scientific (Waltham, USA). 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) was obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against Vimentin, N-cadherin, snail and slug were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against TGF-β receptor I was purchased from abcam (Cambridge, UK). Rhodamine phalloidin was purchased from cytoskeleton (Denver CO, USA). Other reagents were obtained from Sigma-Aldrich (St. Louis, USA).
Cell line and cell culture
Human kidney tubular epithelial cell line HK-2 was obtained from Otwo Biotech (Shenzhen) Inc. (Shenzhen, China).
HK-2 cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China) and 1% penicillin-streptomycin (Gibco, USA) in a humidified incubator with 5% CO2 at 37 oC.
Cell viability assay
The viability of NDQ in HK-2 cells was determined by MTT assay as previously described . Cells (104/well) were seeded in 96-well plates for 24 h and then exposed to different concentrations of NDQ for 24 h, 48 h and 72 h, respectively. After treatment, the supernatant was discarded and 30 ml of MTT solution (5 mg/ml) was added and incubated for another 4 h at 37 oC. And then, the supernatant was discarded and the purple formazan crystals were dissolved in 100 ml of DMSO and the absorbance was measured at 570 nm by a microplate reader (Multiskan FC, Thermo Scientific, USA).
Colony formation assay
HK-2 cells (300/well) were seeded in 6-well plates for 24 h and then treated with NDQ at different concentrations. After incubated for 24 h, cells were washed with phosphate-buffered saline (PBS), and cultured in fresh medium for 10 days. Then, cells were fixed in 75% alcohol at 4 oC and stained with Giemsa dye .
Cell morphological observation
HK-2 cells (105/well) were seeded in 6-well plates and incubated for 24 h. Cells were then serum-starved for 24 h followed by NDQ treatment for 24 h with or without TGF-β1. After that, cells were washed with PBS and morphological changes were observed under a microscope (IX 53, Olympus, Tokyo, Japan).
Cell migration and invasion experiment
For wound healing experiment, HK-2 cells (3´105/well) were seeded in 6-well plates for 24 h and serum-starved for another 24 h. Cells were then scratched in a straight line with 200-μl pipette tips and treated with different concentrations of NDQ in the presence or absence of TGF-β1. Images of HK-2 cells were acquired with a microscope (IX 53, Olympus, Tokyo, Japan) at 0 h, 12 h and 24 h, respectively.
In the Transwell migration assay, HK-2 cells (105/well) were seeded in Transwell chamber with RPMI-1640 medium for 24 h and 400 μl of RPMI-1640 medium containing 10% FBS was added into the bottom chamber. Cells were then incubated with NDQ for 24 h under TGF-β1-stimulated or unstimulated conditions, and chambers were fixed using 75% alcohol before stained with Giemsa dye. After that, cells remaining on the upper surface of the membrane were removed by wiping and the images of migrated cells were obtained under a microscope (IX 53, Olympus, Tokyo, Japan). The invasion assay shared the same method with the Transwell migration assay, the only difference was, matrigel was applied in the Transwell chamber before cells were seeded.
Western blot analysis
HK-2 cells (3×105 dish) were seeded in 60 mm dishes for 24 h and serum-starved for another 24 h. After treated with different concentrations of NDQ for 48 h with or without TGF-β1 stimulation, cells were harvested and washed with cold PBS, and then lysed for 15 min at 4 oC with RIPA buffer (0.1 mM phenylmethanesulfonyl foride, 0.1 mM sodium orthvanadate, 0.1 mM dthiothreitol and phosphatase inhibitor). After centrifugation at 13,500 rpm for 15 min, supernatants were collected as total protein. The protein concentrations were determined by a BCA protein assay kit. The method of Western blotting was performed as previously described . Protein levels were quantified by ImageJ 1.4.3 (National Institutes of Health, USA).
F-actin fluorescence confocal microscopy
HK-2 cells (3×105 dish) were seeded in confocal dishes for 24 h. After incubated in serum-free medium for 24 h, cells were exposed to NDQ for another 24 h in the presence or absence of TGF-β1. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.5% TritonX-100 in PBS. After blocked with 5% bull serum albumin (BSA) for 15 min at room temperature (RT), cells were incubated with 100 μl of rhodamine phalloidin (70 nM) for 1 h at RT. Subsequently, cells were washed and counterstained with 100 μl of DAPI. The fluorescence was observed by confocal microscopy (LSM800, Carl Zeiss, Oberkochen, Germany).
All data were expressed as the means ± SEM and analyzed by Graphpad Prism 5.0 software (Graphpad software Inc., USA). Tukey’s test was used for multiple comparison. The values were considered statistically significant when P < 0.05.