Journal: Nature Communications
Article Title: LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development
Figure Lengend Snippet: Endocrine cell formation requires LSD1 activity during a short window in early pancreatic development. a Schematic of the human embryonic stem cell (hESC) differentiation protocol to the endocrine cell stage (EN) and experimental plan for LSD1 inhibition. b Immunofluorescent staining for pancreatic hormones insulin (INS), glucagon (GCG) and somatostatin (SST) or PDX1 and NKX6.1 in control EN cells compared to EN cells with early (LSD1i early ) and late (LSD1i late ) LSD1 inhibition (representative images, n = 10 independent differentiations). Scale bar, 50 µm. c qRT-PCR analysis for INS , GCG and SST in control, LSD1i early and LSD1i late EN cells. Data are shown as mean ± S.E.M. ( n = 3 replicates from independent differentiations with n = 3 technical replicates per sample; source data are provided as a Source Data file). P = 7.93 e−4, 1.42 e−2, 2.32 e−4, 8.71 e−4, 3.5 e−2, and 1.52 e−3, respectively, Student’s t -test, 2 sided. d Flow cytometry analysis at EN stage for NKX6.1, PDX1 and INS comparing control, LSD1i early and LSD1i late cells. Isotype control for each antibody is shown in red and target protein staining in green. Percentage of cells expressing each protein is indicated (representative experiment, n = 2 independent differentiations). D, day; AA, activin A; ITS, insulin-transferrin-selenium; TGFBi, TGFβ R1 kinase inhibitor; KC, KAAD-cyclopamine; KGF, keratinocyte growth factor; RA, retinoic acid; EGF, epidermal growth factor; ES, human embryonic stem cells; DE, definitive endoderm; GT, primitive gut tube; PP1, early pancreatic progenitors; PP2, late pancreatic progenitors; EN, endocrine cell stage; FSC-A, forward scatter area.
Article Snippet: Other added components included FBS (HyClone), B-27® supplement (Life Technologies), Insulin-Transferrin-Selenium (ITS; Life Technologies), TGFβ R1 kinase inhibitor IV (EMD Bioscience), KAAD-Cyclopamine (KC; Toronto Research Chemicals), and the retinoic receptor agonist TTNPB (RA; Sigma Aldrich).
Techniques: Activity Assay, Inhibition, Staining, Quantitative RT-PCR, Flow Cytometry, Expressing