br Wound healing and transwell assay br Wound
2.4. Wound healing and transwell assay
Wound healing and transwell assay were used to evaluate the in vitro migration ability of NSCLC cells treated with BPS according to the previous study (Chen et al., 2015). For wound healing assay, cells were cultured in 6-well plates to generate the confluent monolayer. Then, cells were scratched and washed with PBS to remove cell debris. The wound closure was recorded by a microscope under 4× magnification at 0 and 24 h after scratch. The migrated distance was calculated and normalized to the initial wound distance at time zero.
The uncoated polycarbonate filters (8 μm pore size, Corning) was used for in vitro migration assay. Cells treated with or without BPS in medium containing 0.1% FBS were seeded in the upper chamber. For the lower chamber, medium containing 10% FBS was added and served as a chemotactic agent. At the end of experiment, migrated cells were fixed in 4% paraformaldehyde, stained with hematoxylin, and counted under upright microscope.
2.5. Western blot analysis
The proteins were extracted by use of 1× RIPA lysis buﬀer (50 mM
Tris–HCl, pH 8.0; 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS). The concentration was measured by use of bicinchoninc HBX41108 (BCA; Sigma-Aldrich Co) method. Samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to a polyvinylidene fluoride (PVDF) mem-brane (Bio-Rad Laboratories, Inc.), and blocked with 5% skim-milk (Bio-Rad Laboratories Inc.). Then primary antibodies against targeted proteins were incubated with the membrane for 2 h at room tempera-ture. After an addition incubation with IgG-horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz Biotechnology), the bands were visualized by using an ECL chemical luminescent system (GenDEPOT).
Total RNAs purification and cDNA generation were conducted by use of TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Foster City, CA, USA) according to the instructions. The RT-PCR was conducted by use of StepOnePlus Real-Time PCR System (Applied Biosystems) and TaqMan Gene Expression Master Mix containing ROX (Applied Biosystems). The primers for targeted genes were: VEGFA forward: 5′-TACCTCCACCAT GCCAAGTGG T-3′ and reverse: 5′-AGGA CGGCTTGAAGATGTAC-3′; IFNG forward: 5′-TCGGTAACTGACTTGAA TGTCCA-3′ and reverse: 5′-TCGCTTCCCTGTTTTAGCTGC-3′; IL2 for-ward: 5′-AGAACTCAAACCTCTGGAGGAAG-3′ and reverse: 5′-GCTGTC TCATCAGCATATTCACAC-3′; IL3 forward: 5′-GACGAGCACGAACCCTC GAT-3′ and reverse: 5′-TGCAGCTTCCCTGCACAG A-3′; IL4 forward:
Fig. 3. BPS activates Smad2/3 in NSCLC cells. (A) A549 or H1299 cells were treated with 10 nM BPS for 12 h, the phosphorylation and total expression of Smad2/3 were measured by western blot analysis (left) and statistically analyzed (right); A549 (B) or H1299 (C) cells were treated with 10 nM BPS for 12 h, the expression of p-Smad2/3 in cytoplasm and nucleus fractions were measured by western blot analysis (left) and statistically analyzed (right); (D) A549 or H1299 cells were treated with 10 nM BPS for 12 h, the expression of p21 and p27 were measured by western blot analysis (left) and statistically analyzed (right). Data are presented as means ± SD of three independent experiments. **p < .01 compared with control.
5′-CCGTAACAGACATCTTTGCTGCC-3′ and reverse: 5′-GAGTGTCCTTC
TCATGGTGGCT-3′; IL5 forward: 5′-GGCACTGCTTTCTACTCATCGA-3′
and reverse: 5′-AGTTGGTGATTTTTATGTACGGAACA-3′; IL6 forward:
5′-CCTCCAGAACAGATTTGAGAGTAGT-3′ and reverse: 5′-GGGTCAGG
GGTGGTTATT GC-3′; IL8 forward: 5′-AAGACATACTCCAAACCTTTCC
forward: 5′-AACCTGCCTAACATGCTTCGA-3′ and reverse: 5′-CTCATG
GCTTTGTAGATGCCT-3′; EGF forward: 5′-TGGATGTGCTTGATAAG
ward: 5′-GGCCAGATCCTGTCCAAGC-3′ and reverse: 5′-GTGGGTTTCC
ACCATTAGCAC-3′;GAPDH forward: 5′-ATGTTCGTCATGGGTGTGAA
lative expression levels of genes were normalized to GAPDH by use of 2− Ct method.
The expression of TGF-β in medium was measured by use of TGF-β ELISA kit (R&D Systems) according to the manufacturer's instructions (R&D).
2.8. Statistical analysis
Data was analyzed by GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego CA). Diﬀerences between two groups were assessed using the unpaired Student's t-test. Results with P < .05 were considered statistically significant.
3.1. Nanomolar BPS can trigger the migration of NSCLC cells
To evaluate the eﬀects of BPS on the biological functions of NSCLC
cells, we treated cells with 1, 10, 100 nM of BPS, which covered the concentration ranges measured in human body. Our data showed that 1, 10, 100 nM of BPS had no significant eﬀect on proliferation of A549, H1299, or H358 cells (Fig. 1 A). Wound healing assay showed that both 1 and 10 nM can significantly increase the in vitro migration of A549 cells (Fig. 1 B). Similarly, BPS also triggered the migration of H1299 cells (Fig. 1 C). The promotion eﬀects of 10 nM BPS on in vitro mi-gration of NSLC cells were further confirmed by transwell assay (Fig. 1 D). Among the measured cell motility related markers, BPS can increase the expression of MMP-2 and vimentin in both A549 and H1299 cells (Fig. 1 E).