Transwell chamber

Transwell assay was used to examine the effect of CuB on the cell migration process. A549 cells were starved for 1 h, then used 1% FBS culture medium with TGF-β1 (4 ng/mL) in the presence or absence of CuB (5, 10, 15 nM) on the upper region of the transwell chamber (8-μm pore size, Thermo Fisher). The lower chamber of the transwell was added with 500 μL 10% FBS culture medium. A549-GR cells were starved for 1 h, then used 1% FBS culture medium with CuB (15 nM) or Gefitinib (10 μM) on the upper region of the transwell chamber (8-μm pore size, Thermo Fisher). The lower chamber of the transwell was added with 500 μL 10% FBS culture medium. The cells after incubation at 5% CO₂ and 37 ℃ for 48 h, then fixed with 4% paraformaldehyde (PFA) for 15 min, washed with PBS for 3 times, and then stained with crystal violet for 10 min, the cells within upper transwell chamber were removed with a cotton swab. The migration cells were imaged by using a microscope, and the relative proportion was calculated with Image J.
Matrigel transwell invasion assay was performed to detect the inhibition effect of CuB on the cell invasion process. Briefly, Matrigel was diluted with 1% FBS culture medium at a ratio of 1:12, and added 100 μL to the transwell chamber, incubation at 5% CO₂ and 37 ℃ for 1 h. Then the other steps are consistent with the transfer experiment.

For migration assay, exuberant cells were harvested and resuspension in RPMI-1640 medium (Gibco) supplemented with 2% FBS. 400 μL suspension medium containing 1.2 × 10⁵ cells were added into the transwell chamber (Corning), and 500 μL culture medium with 10% FBS was added into the lower chamber of the 24-wells plate. After cultured for 18 h, the transwell chamber was fixed with 4% paraformaldehyde for 20 min, followed by stained with crystal violet for 30 min. Then transwell chamber was gently washed and air-dried. Cells on the membrane was counted with a microscope.
For invasion assay, Matrigel matrix (#354248) was used to simulate the extracellular matrix environment. Briefly, Matrigel matrix that thawed at 4 °C overnight was diluted with pre-cold opti-MEM (Gibco) at the ratio of 1:3, 165 μL diluted Matrigel matrix was added into the transwell chamber and placed in the incubator at 37 °C for 30 min. The unbounded Matrigel matrix was gently removed. Exuberant cells were harvested and resuspended to the concentration of 1.5 × 10⁵ cells per 400 μL using RPMI-1640 medium containing 2% FBS. 400 μL suspension medium was added into the chamber and the lower compartment was filled with 500 μL medium containing 10% FBS. After cultured at 37 °C for 24 h, cells on the membrane of the transwell chamber was fixed, stained and then observed and counted under the microscope.

The compound paeonol (99% purity) was obtained from Baicao Plants Biotech Co., Ltd. (Anhui, China). Dulbecco's modified Eagle's medium (DMEM), Transwell chamber, type I collagenase, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies, Co., Ltd. (Paisley, UK). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Lactic dehydrogenase (LDH) reagent was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against PDGF-B, Ras, Raf, phosphorylated Raf (P-Raf), ERK1/2, and phosphorylated ERK1/2 (P-ERK1/2) were obtained from Cell Signaling Technology (Beverly, MA, USA). PDGFR inhibitor (Sunitinib Malate) and ERK1/2 inhibitor (PD98059) were purchased from Santa Cruz Biotechnology Co. (Santa Cruz, CA, USA).