Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: A disrupted expression of ectoderm-associated genes in embryonic bodies spontaneously differentiated from IFNB-iPSCs. IFNB-iPSC lines LA8 and LC8 and parental K7-iPSCs were cultured in low-adhesive conditions to stimulate the formation of embryoid bodies (EBs). On day 20, RNA was isolated and gene expressions were analyzed using RT-PCR. Whole-cell lysates of EBs were also analyzed by a Western blot. ( a – c ) Relative expressions of endoderm- ( a ), mesoderm- ( b ), and ectoderm- ( c ) associated markers in iPSCs and 20-day EBs. Data are shown as boxes and whiskers with minimal and maximal values (summarized results of at least 2 independent experiments). The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Figures indicate the FDRs for the main comparisons, irrespectively of their significance (i.e., inter-line comparisons on differentiation day 20 and intra-line comparisons between iPSCs and 20-day EBs). ( d ) The Western blot analysis of PAX6 protein in IFNB-EBs and K7-EBs (one experiment). The graph shows the relative densitometric values of PAX6 normalized to HSP90.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Adhesive, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot