Table 1.
Primers used in this study for RT-PCR or real time PCR.
Table 2.
Primers used for CDH2 ChiP assay.
Figure 1.
Knocked down SMAD4 reduces N-cadherin protein level and inhibits invasion and migration in HPNE cells.
(a) Western blot analysis of cell expression of SMAD4 and N-cadherin and CK19. Actin was used as the loading control. (b) RT-PCR results showing cell mRNA levels of CDH1, CDH2, CK19, VIM, BCAT, and FN1, and (c) TWIST1, TWIST2, SNAIL2, and ZEB1. GAPDH was used as the housekeeping control. (d) Immunofluorescence staining of CK19 and N-cadherin in HPNE/shScr or HPNE/shSMAD4 cells. CK19 and N-cadherin were labeled with red fluorescent Alexa Fluor 594 goat anti-rabbit IgG (A11012, Invitrogen). GFP-positive cells represent cells transfected with shScr or shSMAD4. Nuclei were counterstained with blue fluorescent 4,6-diamidino-2-phenylindole. Images were merged using Olympus CellSens software. (e) Modified Boyden chamber assay. HPNE/shScr and HPNE/shSMAD4 Cells were added with or without 10 ng/ml TGF-β to serum-free media inserts in the top chamber, and 20% FBS was placed in the bottom chamber as a chemoattractant. Invasive cells were counted in 3 fields at 10× magnification in duplicated inserts. (f) Wound-healing assay. HPNE/shScr and HPNE/shSMAD4 cells were treated with or without10 ng/ml TGF-β. The y-axis represents cell migration distance at the time of the scratch and after 20 hours. Three random images (4×) were taken at these time points, and migration rate was determined as the ratio of distance at 20 hours versus 0 hours in the wound's gap using Adobe Photoshop software. Results are the mean ± s.d. of 3 independent experiments.
Figure 2.
N-cadherin alteration after TGF-β treatment in HPNE, HPNE/shScr, and shSMAD4 cells.
(a) Western blot analysis of cellular expression levels of N-cadherin and SMAD4 in cells treated with TGF-β (5 ng/ml) for 2 hours, 8 hours, 24 hours, or 5 days. Actin was used as the loading control. (b) CDH2 mRNA level was measured by RT-PCR after TGF-β treatment (5 ng/ml) in 2 hours, 8 hours, and 24 hours' time points. GAPDH was used as the housekeeping gene control.
Figure 3.
Map of multiple SBEs in CDH2 promoter.
(a) Three SBEs with a CAGACA sequence (blue circles 1, 2, and 3) and one SBE with a GTCTAGAC sequence (red circle 4). Sections A, B, and C represent 3 primers and an amplifying region for ChIP assay in the promoter. (b) Electrophoretic mobility shift assay results showed that 4 SBE oligos had strong DNA and nuclear protein interaction bands (lane 1), binding was quenched by wild-type (WT) oligos (lane 2) and not by mutant (M) oligos (lane 3), and anti-SMAD4 antibody (Ab) inhibited binding activity (lane 4). (c) SBE binding activity was regulated by TGF-β treatment at 0, 2, 8, and 24 hours. Oct-1 DNA binding activities were determined as loading controls in (b) and (c). (d-f) ChIP assays and real-time PCR of primers A, B, and C comparing the ratio of IgG to anti-SMAD4 antibody with or without 5 ng of TGF-β at 0 2, 8, and 24 hours. *P<0.05, **P<0.01, ***P<0.001.
Table 3.
Oligos from CDH2 promoter for EMSA.
Figure 4.
TGF-β Stimulated CDH2 promoter Dual-luciferase Activity.
(a) Schematic of five pGL2-CDH2 promoter constructs, including part of the CDH first exon (white rectangles), and potential SBE sites (downward arrows). (b,c) Luciferase activity ratios of CDH2 clone #5 and F1/R1 construct treated with or without 5 ng of TGF-β at 0, 2, 8, and 24 hours in HPNE and 293T cells. **P<0.01, ***P<0.001. (d) The second SBE sequence in the CDH2 promoter and the Mut2 mutant sequence (G→C). (e) Luciferase activity ratio of Mut2 with or without 5 ng TGF-β at 0, 2, 8, and 24 hours in HPNE and 293T cells.
Figure 5.
Invasion and migration assay after CDH2 knockdown in HPNE, HPNE/shScr, and HPNE/shCDH2 cells.
(a) N-cadherin was suppressed though shCDH2. Actin was used as the loading control. (b) RT-PCR (left) and real-time PCR (right) confirmed that CDH2 mRNA was significantly decreased in HPNE shCDH2 cells. (c) Modified Boyden chamber assay was performed with transfected cells. Cells were treated with or without 10 ng/ml TGF-β in serum-free medium in the top inserts, and 20% FBS medium was used in the bottom chamber as a chemoattractant. Invasive cells were counted in 3 fields at 10× magnification in duplicated inserts. (d) Wound-healing assay was performed with transfected cells treated with or without 10 ng/ml TGF-β. Three random images (4× magnification) were taken at the time of the scratch (0 hours) and at 20 hours. Migration rate was determined as the ratio of the distance traveled at 20 hours versus 0 hours in the wound's gap using Adobe Photoshop software. **P<0.01, ***P<0.001.
Figure 6.
SMAD4 and N-cadherin expression levels in patient tumors and matched xenograft samples.
(a) Representative immunostained images of patients' primary tumors and (b) and matched patient-direct xenograft tumors (PATX). Images were captured with an Olympus DP72 camera (10× magnification). (c) Western blotting results for corresponding patient-direct xenograft tumor lysates. Actin was used as the loading control.