Vimentin

Western blotting was performed as described previously [19 (link)]. The following primary antibodies were used: LKB1 (ab15095, 1:100) from Abcam (Cambridge, UK); E-cadherin (BS1097, 1:500), vimentin (BS1776, 1:500), β-actin (BS6007 M, 1:10,000), p-CHK2 (p-T68) (BS4043, 1:500), and γ-H2AX (p-S139) (BS4760, 1:500) from Bioworld Technology (Nanjing, Jiangsu, China); and SIK1 (51045-1-AP, 1:1000) and ZEB1 (21544-1-AP, 1:1000) from Proteintech Group (Wuhan, Hubei, China). All primary antibodies were incubated with the blot at 4°C overnight. The signals were detected with an Odyssey Infrared Imaging system (LI-COR, Lincoln, NE, USA). For quantification of the protein levels, the intensity of each strip was analyzed by Image J software (NIH, Bethesda, MD, USA). The average intensities of the proteins were normalized to β-actin. The relative protein levels are presented as mean ± standard deviation (SD).

Briefly, after deparaffinization and hydration, the slides were inactivated endogenous peroxidase by using 3% hydrogen peroxide and heat-pretreated in ethylene diamine tetraacetic acid (PH 8.0) by a microwave oven for 5 min. Then, anti-KIF3B (Abcam, USA, ab152976, 37°C, 2h, 1:200) and secondary antibody (37°C, 30 min) were incubated. Sections were stained with Diaminobenzidine (DAB) and counterstained with hematoxylin. PBS was used as negative control. Other antibodies were as follows: β-catenin, CyclinD1, C-myc, p-GSK-3β^ser9, E-cad, Vimentin, MMP-2 and MMP-9 (Bioworld Technology, all at dilution 1:200), Dvl2, MMP-7, Slug and Snail (Bioss, all at dilution 1:200). Independent Histologic and IHC evaluation were conducted by two pathologists. IHC staining of KIF3B was scored as described previously (43 (link), 44 (link)). The scores of staining intensity (0, none; 1, weak; 2, intermediate; and 3, strong) and positive tumor cell proportion (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, > 2/3) were summed up to obtain a total score (ranging from 0 to 8).

Nicotine-treated hUC-MSCs were fixed in 4% paraformaldehyde for 30 minutes, permeabilized for 10 minutes with 0.1% Triton-X 100 blocked with 5% bovine serum albumin (BSA), and incubated with rabbit monoclonal rabbit monoclonal anti-N-cadherin (1:200), Vimentin (1:100), β-catenin (1:200) and FAP (1:500) (Bioworld Technology) antibodies overnight at 4°C, followed by incubation with Cy3-labeled anti-rabbit IgG secondary antibody (1:800) at 37°C for 45 minutes in the dark. Finally, the nuclei were counterstained with Hoechst 33342 (1:200; Sigma-Aldrich). Fluorescent images were acquired at 200× using an inverted fluorescence microscope (Ti-S, Nikon).

Baicalin with 98% purity was purchased from National Institute for the Control of Pharmaceutical and Biological Product (Hangzhou, China); 5-FU was obtained from Yuanye Biological Technology (Shanghai, China). Baicalin and 5-FU were dissolved in dimethyl sulfoxide (DMSO). TGF-β1 was purchased from PeproTech and treated cells for 12 h in this study. Antibodies for Smad3, p-Smad3, Smad2, p-Smad2 and Smad4 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies for Smad7, Akt, p-Akt, Cyclin B1, Cyclin D1, P21, P53, Parp-1, Caspase 3, XIAP, Survivin and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA. USA). Antibodies for CD133, CD44, Nanog, OCT4, SOX2, Bcl-2, Bax, P27, Caspase8, Caspase9, Snail, Twist and Slug were obtained from Proteintech (Rosemont, IL, USA). Antibodies for TGF-β1, N-Cadherin, E-Cadherin, Vimentin, Cytokeratin 18, Claudin 1, NF-κB-p65 and Cyclin E1 were purchased from Bioworld Technology Inc. (St Louis, MN, USA).

Antibodies specific for c-Myc (Cell Signaling Technology, Beverly, MA) and β-actin (Sigma, St. Louis, MO) were used for Western blot analysis. For immunostaining analysis, antibodies specific for the following proteins were used: ER, PR, SMA, and K5 (Novus Biologicals, Littleton, CO); K14, vimentin, and p63 (Bioworld Technology, Louis Park, MN); K8 (Abcam, Cambridge, MA); c-Myc (9E10) and HA probe (Santa Cruz Biotechnology, Santa Cruz, CA); claudin 3 and claudin 4 (Invitrogen). An antibody specific for Id1 (C-20, Santa Cruz Biotechnology) were used for both Western blot and immunostaining analysis.