Journal: Scientific Reports
Article Title: Human esophageal myofibroblast secretion of bone morphogenetic proteins and GREMLIN1 and paracrine regulation of squamous epithelial growth
doi: 10.1038/s41598-018-30799-7
Figure Lengend Snippet: HEMF secreted factors modulate BMP signaling in squamous epithelial cells. ( a ) pSMAD1/5/8 immunofluorescent staining (Cy2, green) was performed on paraffin-embedded, formalin-fixed sections of 3D organotypic ALI culture established with control serum-free myofibroblast media (SFMM), conditioned SFMM (cSFMM), and cSFMM plus the addition of GREM1 nAb. Representative images of three independent experiments is shown. Images were viewed with a confocal microscope. Magnification 40x. ( b ) Immunoblots for pSMAD1/5/8 and ID1 were performed on epithelium harvested from 3D organotypic ALI culture. Epithelial pSMAD1/5/8 and ID1 increase in the presence of cSFMM. Addition of GREM1 nAb inhibits the increase in pSMAD1/5/8 and ID1. Tubulin was used as the loading control. Representative immunoblots are shown of three individual experiments. Samples from each experiment were derived at the same time and processed in parallel. Relative densitometry to loading control tubulin is shown. *P ≤ 0.001; **P ≤ 0.02; **P ≤ 0.05; NS, non-significant with ANOVA for multiple comparisons.
Article Snippet: The membrane was blocked with 5% milk or BSA and incubated overnight with rabbit anti-p63 (0.33 μg/ml, Genetex, #GTX102425), mouse anti-PCNA (1:400, BD Transduction, cat#610664), rabbit anti-pSMAD1/5/8 monoclonal (1:500, Cell Signaling, cat# 9516), rabbit anti-SMAD5 monoclonal (1:1000, Cell Signaling, cat# 12534 S), or rabbit anti-ID1 polyclonal antibody (0.5 μg/ml, Santa Cruz #sc-488).
Techniques: Staining, Control, Microscopy, Western Blot, Derivative Assay