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: IFNB-iPSCs express functional IFN-β. IFNB-iPSCs and K7-iPSCs were cultured in parallel and used for RNA isolation; the preparation of cell extracts; and the collection of cell culture supernatants. ( a ) IFNB-iPSCs display an increased expression of IFNB as compared with the parental K7-iPSC line. RNAs isolated from IFNB-iPSCs and K7-iPSCs were subjected to RT-PCR; IFNB expression values were normalized relative to GAPDH , and fold changes were calculated as relative IFNB mRNA levels in IFNB-iPSCs relative to K7-iPSCs (2 −∆∆Cq ). Dotted line, fold change = 2 (significance threshold). Data are shown as boxes and whiskers with minimal and maximal values (K7-iPSCs, LA8-iPSCs, and LC8-iPSCs, summarized results of at least 3 independent experiments; LE4-iPSCs, one experiment, technical replicates). Note that LA8-iPSCs and LC8-iPSCs bear a homozygous IFNB insertion, whereas LE4-iPSCs are heterozygous. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( b , c ) IFNB-iPSCs express IFN-β at the protein level. ( b ) IFNB-iPSCs and K7-iPSCs were pelleted, frozen, and analyzed in a Western blot. The graph shows the relative densitometric values of IFN-β normalized to β-actin. ( c ) The supernatants were collected from IFNB-iPSCs and K7-iPSCs and analyzed using ELISA (the results of two independent experiments; in each of them, K7-iPSCs, LA8-iPSCs, and LC8-iPSCs were cultured in parallel and independently of cells of another experiment). ( d ) The supernatants of IFNB-iPSCs exhibit IFN-β-like functional activity. The supernatants were obtained from IFNB-iPSC and K7-iPSC cultures and were added to THP-1 macrophage-like cells pre-activated with PMA. Control THP-1 cells were cultured in the absence of iPSC supernatants. THP-1 RNA was isolated from all cultures, and the expressions of ISGs were analyzed in RT-qPCR using RPL27 as a housekeeping gene. Data are shown as boxes and whiskers with minimal and maximal values and individual points. The representative results of one out of two experiments are presented. Numbers on the graphs show the FDRs (Benjamini, Krieger, and Yekutieli correction for multiple comparisons). FDRs < 0.05 were considered as significant; for the comparison of IFNB-iPSCs and K7-iPSCs, FDRs > 0.05 are also shown. The differences between LA8-iPSCs and LC8-iPSCs were insignificant. PMA, phorbol 12-myristate-13-acetate.
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: Functional Assay, Cell Culture, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Quantitative RT-PCR, Comparison
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