Table 1.
Patient characteristics of biopsies used for Western blot, IHC and Q-RT-PCR analysis.
Table 2.
Primer sequences.
Table 3.
Antibodies used for Western blot analysis.
Fig 1.
Expression of BMP4, BMP4 pathway associated molecules and the downstream target ID2.
a. Q-RT-PCR was used to determine mRNA expression of BMP4, its downstream target, ID2, BMP4 associated receptors and SMAD molecules in SQ, BE and EAC biopsy specimens. B2M and GAPDH were used for normalization. Data are relative to the mean ΔCt of SQ biopsies and are expressed as box plots, representing the mean with the minimum and maximum values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. b. Western blot analysis in SQ, BE and EAC biopsy specimens showed BMP4, and ID2 expression and phosphorylation of SMAD1/5/8 in BE and EAC. Actin was used as loading control. Biopsy samples from 6 EAC patients were used. Representative pictures are shown. c. IHC showed nuclear and cytoplasmic expression of SMAD4 in 10 of 13 BE (arrowhead) and 11 out of 13 EAC tissue sections. EAC* represents a biopsy specimen with positive SMAD4 staining, EAC** represents a biopsy specimen with negative SMAD4 staining, stromal cells are SMAD4 positive and serve as internal control. Haematoxylin counterstain was used. Representative pictures are shown.
Fig 2.
ID2 expression upon BMP4 or Noggin incubation.
a. mRNA expression of the downstream target ID2. B2M was used for normalization. Data are relative to the mean ΔCt of control cells and are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. b. Western blot analysis of BAR-T and OE33 cells incubated with BMP4 showed upregulated ID2 expression and more phosphorylation of SMAD1/5/8. Cells incubated with Noggin showed downregulated ID2 expression. Actin was used as loading control.
Fig 3.
Migration assays of BAR-T and OE33 cells upon incubation with BMP4 or Noggin.
a. In vitro scratch assay of BAR-T and OE33 cells. The rate of migration across the scratched area was monitored for 24 h. Representative images showed that the scratch induced cells incubated with BMP4 to migrate compared to control cells or cells incubated with Noggin. Images are representative of three independent experiments. b. Boyden chamber assay of BAR-T cells incubated with BMP4 or Noggin. Data are relative to control cells not incubated with BMP4 or Noggin and expressed as mean ±SD. * p<0.05, ** p<0.01.
Fig 4.
mRNA and protein expression of factors associated with EMT upon BMP4 or Noggin incubation.
a. Q-RT-PCR was performed to determine mRNA expression of SNAIL2 and its target genes, CDH1, CDH2 and Vimentin. B2M was used for normalization. Data are relative to the mean ΔCt of cells incubated with Noggin and are expressed as mean±SEM. *p<0.05. b. Western blot analysis of BAR-T and OE33 cells showed that SNAIL2 expression was upregulated and CDH1 expression was downregulated in cells incubated with BMP4 compared to cells incubated with Noggin. Actin was used as loading control. Pictures are representative of three independent experiments.