NSCLC NCI-H1299 and A549 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37̊C in 5% CO₂. Both cell lines were presented as a gift from Dr Yongguang Tao from the Cancer Research Institute of Central South University. NCI-H1299 was confirmed to be a Taxol-resistant cell line with high expression of Op18/stathmin in our previous study (8).

Primary antibodies included rabbit polyclonal anti-stathmin (Calbiochem, La Jolla, CA, USA), rabbit monoclonal anti-stathmin (phospho-Ser16, -Ser25, -Ser38 and -Ser63) (Abcam, Cambridge, MA, USA), mouse monoclonal anti-caspase 8, rabbit monoclonal anti-caspase 9, rabbit monoclonal anti-PP2A C subunit (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphoserine (Invitrogen, Carlsbad, CA, USA), part antibodies including rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-NF-κB, mouse monoclonal anti β-actin, mouse monoclonal anti-caspase 3, mouse monoclonal anti-interleukin-10 (IL-10), which were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Secondary antibodies included rabbit anti-mouse HRP-conjugated IgG and goat anti-rabbit HRP-conjugated IgG (Santa Cruz Biotechnology, Inc.).

Taxol (sc-201439) and VBL (sc-491749) were also obtained from Santa Cruz Biotechnology, Inc., and were dissolved in dimethyl sulfoxide (DMSO) at appropriate concentrations, and were stored at −20°C.

Upon reaching 80% confluency, the cells were treated with Taxol or VBL (100 nM) for 24 h, and the solvent DMSO was used as control. Then, the treated cells were harvested and stained with 500 µl specific binding buffer containing 5 µl Annexin V-FITC and 5 µl propidium iodide for 10 min at room temperature in the dark. Analysis of cell apoptosis was performed by flow cytometry at the The Second Xiangya Hospital Affiliated with Central South University.

Cells (5,000/well) in a logarithmic growth phase were planted into 96-well plates and treated with Taxol or VBL (100 nM). Next, 10 µl of 5 mg/ml 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to each well at time points of 0, 24, 48 and 72 h, and the cells were subsequently incubated at 37̊C for 4 h. After the culture supernatants were removed, 100 µl DMSO was added for 10 min, and the absorbance was measured at a 490-nm wavelength. Six parallel wells were performed.

Cellular proliferative viability was calculated using the following formulation: Relative proliferative percentages (%) = (OD_treatment/OD_control) × 100%. The percentage of the DMSO control was designed as 100% at all time points.

Cells (2,000/well) were cultured in media with 10 nM Taxol or VBL in 6-well plates for 1–2 weeks. The culture was suspended when obvious colonies were observed by the naked eye. Cells were fixed with methanol, and then stained with 0.1% crystal violet for 30 min. Colonies with >50 cells/colony were counted using an inverted microscope. The experiment was carried out in triplicate independently. The relative colony formation efficiency (%) = (colony number/2,000) × 100%.

Cells were grown in 6-well plates to a monolayer. The cell monolayer was then wounded by creating a scratch using a 200-µl pipette tip. Old medium was replaced with fresh medium containing 100 nM Taxol or VBL. The status of wound healing was monitored at time points of 0, 12, 24 and 36 h, and images were captured for assaying the capability of cell migration in a 2-dimensional plane.

Capability of cell migration in a 3-dimensional space was assessed by Transwell assays according to the protocol described in a previous study (8). Approximately 4×10⁴ cells were seeded in 200 µl RPMI-1640 medium with 0.2% FBS in the upper chambers of a Transwell, while 800 µl media with 10% FBS was placed in the lower chambers. Twenty-four hours later, the media were removed and the cells were wiped off from the upper chambers using a cotton swab. The remaining migrated cells on the back of the bottom membrane in the upper chamber were fixed with 100% methanol and stained with 0.1% crystal violet. Finally, the Transwell plates were mounted and observed under an inverted microscope, and the migrated cells were counted and images were captured.

Protein extracts were collected according to our previous studies (9,10). Briefly, cells were lysed with lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 2% SDS, 5 mM DTT, 10 mM PMSF), and then denatured in boiling water for sonication. Protein concentrations were determined using BCA protein assay reagent (Pierce, Rockford, IL, USA).

Protein extracts (50 µg) were separated by 10% SDS-PAGE, electrotransferred to nitrocellulose membrane, blocked with buffer containing 5% non-fat milk and incubated with primary antibodies overnight at 4̊C and HRP-conjugated secondary antibodies for 1 h at room temperature and developed with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were lysed in immunoprecipitation (IP) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10% NP-40, 1 mM EDTA, 10% glycerol, 10 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail tablet), and then the supernatant was collected for detection of the total level of phospho-Op18/stathmin.

Magnetic immunobeads (cat#: 10002D; Thermo Fisher Scientific) were used to bind the anti-stathmin antibody and pull-down Op18/stathmin for western blot analysis as previously described (9).

Equal number of cells were planted in 6-well plates and cultured. The old medium was replaced with fresh medium without phenol red indicator in the presence of Taxol or VBL (100 nM) when cells reached 80% confluency. Twenty-four hours later, the media were collected and centrifuged at 13,000 rpm for 30 min. Autocrine IL-10 in the separated supernatant was detected by ELISA using the human IL-10 ELISA kit (Cusabio, Wuhan, China). All experiments were carried out in triplicate.

Plasmids pGCsilencer-U6/Neo/GFP-RNAi (RNAi) with the RNAi gene targeting the coding region of Op18/stathmin at 5′-AGAGAAACTGACCCACAAA-3′ (GenBank no. 53305, sequence 374-393) and pGC-silencer-U6/Neo/GFP-non (Non) with an inserted non-coding sequence and pGC-silencer-U6/Neo/GFP (Blank) without any fragments inserted, were constructed at an early stage and were introduced into cells by Lipofectamine 2000 (Invitrogen) as described in our previous studies (8,10).

All statistical calculations were performed using the statistical software program SPSS 17.0. Differences with p-values <0.05 were considered statistically significant.

Morphological images showed that cells grew to nearly complete confluency in the control DMSO, whereas cell distribution became gradually dispersed with the appearance of a few translucent floating cells in the Taxol treatment cell group. A large number of suspended cells emerged and only a minority of cells adhered to the wall at a low density in the VBL treatment group (Fig. 1A).

Cell apoptotic ratios were 8.65, 14.95 and 22.66% following treatment with the solvent (DMSO), Taxol and VBL, respectively, in the NCI-H1299 cells as detected by FCM. The apoptotic ratio was significantly higher for VBL treatment compared with Taxol (Fig. 1B).

Western blot analysis showed that both Taxol and VBL increased the expression levels of caspase 3 and 9, but to a greater extent following VBL treatment, while no obvious changes in caspase 8 were observed in the three treatment groups (Fig. 1C).

VBL significantly inhibits cell proliferation and colony formation compared with Taxol. By contrast with DMSO, the relative cell proliferation percentages were 84.24, 55.11 and 32.00%, respectively, for VBL treatment at time points of 24, 48 and 72 h, while these percentages were 95.86, 91.47 and 86.46%, respectively, following Taxol treatment. The representative curve steeply declined with increasing exposure time to VBL, whereas, it declined slowly in the presence of Taxol (Fig. 2A). Statistical differences existed in the proliferation percentages between VBL and that of the other two groups for Taxol and DMSO at 24, 48 and 72 h (p<0.01) (Fig. 2B).

Colony formation assays revealed that only a few small colonies appeared in the VBL treatment group, while some relative larger colonies emerged in the Taxol treatment group. There were a large number of large colonies which generally merged into an extensive one with unclear borders in the control DMSO group (Fig. 2C). The mean colony formation efficiency was 11.60, 9.47 and 1.07%, respectively, in the DMSO, Taxol and VBL treatment group. VBL significantly reduced the percentage of colony formation in comparison with the Taxol and DMSO treatment groups (p<0.05) (Fig. 2D).

The scratch-wound trails obviously became narrow with time in the three groups, and were completely recovered at time point of 36 h in the DMSO treatment group, while the scratched areas nearly healed for Taxol induction. However, a large area of blank region still remained in the cells treated with VBL (Fig. 3A).

Transwell assay analysis revealed that the mean number of invading cells was 27, 16 and 8, respectively, in the DMSO, Taxol and VBL treatment group. The number of migrated cells was greatly decreased following treatment with VBL when compared to the Taxol treatment group, whereas Taxol also inhibited cell migration in contrast with DMSO (Fig. 3B). Differences in the number of migrating cells were significant between the VBL group and the other two treatment groups; VBL was the most notable in inhibiting cell migration of a 3-dimensional space (p<0.01) (Fig. 3C).

Western blot analysis revealed that the expression of Op18/stathmin was decreased by Taxol, but was not obviously affected by VBL (Fig. 4A). IP-western blotting showed that Taxol decreased the phosphorylation of Op18/stathmin; however, VBL upregulated the total level of phosphorylated Op18/stathmin (Fig. 4B).

VBL universally increased the level of phosphorylated Op18/stathmin at all 4 serine sites including Ser63, Ser38, Ser25 and Ser16, while Taxol mainly reduced phosphorylated Op18/stathmin at Ser25 and Ser63 sites, particularly, p-Ser63-Op18/stathmin was almost completely inhibited, yet Taxol did not influence the phosphorylation of Op18/stathmin at Ser16 and Ser38 (Fig. 4C).

MTT analysis demonstrated that the proliferation ratios were 99.7, 96.79 and 94.48%, and 68.73, 54.38 and 50.88%, at time points 24, 48 and 72 h in the non-silenced and the RNAi groups respectively in comparison with the DMSO group. The representative curve of the RNAi group declined rapidly, the curve of the non-silenced group nearly overlapped the one of the Blank. After co-treatment with Op18/stathmin RNAi and VBL or Taxol, the relative proliferative ratios were 60.95, 40.32 and 27.16%, and 58.69, 33.20 and 24.66%, respectively, at the three time points, while the corresponding curves were highly similar between VBL and Taxol, which declined steeply compared with the one for single RNAi (Fig. 5A). Histograms showed that the difference was not significant between the Blank and the non-silenced group, but was increased between the RNAi groups and the blank control (p<0.01). The combination of RNAi and VBL or Taxol significantly inhibited cell proliferation in contrast with RNAi alone treatment (p<0.01), whereas Op18/stathmin RNAi reduced the difference in cell proliferation inhibition between VBL and Taxol at all time points (Fig. 5B).

Cell growth images show that cell proliferation was obviously inhibited by RNAi, while the inhibition was increased following co-treatment of RNAi and VBL or Taxol. Cells became much sparser in the co-treated groups with RNAi than the RNAi alone treatment group (Fig. 5C).

Western blot analysis showed that Op18/stathmin RNAi effectively inhibited the expression of Op18/stathmin in the NCI-H1299 cells (Fig. 5D).

Western blot analysis showed that Taxol notably enhanced the expression of protein phosphatase 2A (PP2A), but PP2A was substantially decreased by VBL, while both Taxol and VBL treatments decreased the expression of nuclear factor-κB (NF-κB), B-cell lymphoma-2 (Bcl-2). However, the inhibitory effects were more notable for VBL induction than Taxol (Fig. 6A).

ELISA demonstrated that autocrine IL-10 from cell culture supernatants was decreased by Taxol and VBL, but VBL induced more obvious inhibitory effects when compared with Taxol. Histograms showed that the differences were significant between the VBL and the other two treatment groups (Fig. 6B).

Western blot analysis showed that both Taxol and VBL inhibited the expression of IL-10 in the NCI-H1299 cells, but VBL was more effective than Taxol (Fig. 6C).

FACS showed that cellular apoptotic percentages were 6.07, 13.47 and 21.81%, respectively, in the NCI-H1299 cells in the DMSO, Taxol and VBL treatment groups, while they were 6.95, 21.59 and 19.21% respectively in A549 cells in the corresponding groups. NCI-H1299 cells presented notable resistance to Taxol when compared with VBL, but the sensitivity of Taxol was similar to VBL in the A549 cells (Fig. 7A).

Western blot analysis demonstrated that NCI-H1299 cells highly expressed Op18/stathmin compared with the A549 cells (Fig. 7B).