Journal: NPJ Aging and Mechanisms of Disease
Article Title: Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells
Figure Lengend Snippet: SA-miRNAs from oncogenic MIR17HG and tumor-suppressive MIR100HG clusters function to establish senescent phenotype in hADSCs. a Percentage of SA-β-Gal positive cells among the total amount of cells counted after transient transfection of the mimics of the SA-miRNAs from either the MIR17HG ( mir-17-5p , mir-18a-5p , mir-19a-3p , mir-20a-5p and mir-92a1-5p ) or the MIR100HG ( mir-125b1-5p , mir-1let7a-2-3p , mir-100-5p ) clusters separately or after simultaneous transfection by a full set of the SA-miRNA mimics from both clusters in SR hADSCs. SA-miRNA mimics were transfected with FITC-labeled control to account for the transfection efficiency as described in material and methods and electronic supplementary materials . The transfection efficiency for each combination is shown in (Supplementary Figure S8D ) and is expressed as a percentage of green cells among the total DAPI-positive cells ( n ) counted under the fluorescent microscope. b Quantitation of the BrdU incorporation in SA-miRNA mimics-transfected SR hADSCs (FITC+) 72 hrs post-transfection presented as FITC+ BrdU+ ( red bar ). Quantification of cells positive for SEN-associated markers P21 WAF1/Cip1 and γH2AX persistent DDR focal staining is performed under the same conditions in FITC+ ( red bars ) and FITC− cells. Results were plotted on the graphs as the averages of three independent experiments (biological replicates n = 3) with the standard deviation of data. Cell counted in each experiments: BrdU staining n 1 = 440, n 2 = 279, n 3 = 334; γH2AX staining n 1 = 117, n 2 = 109, n 3 = 133; P21 WAF1/Cip1 staining n 1 = 118, n 2 = 135, n 3 = 90. P -values (p) were calculated as Students’ two-tailed test: BrdU labeling experiment ***p
Article Snippet: Total RNA and miRNA was reverse transcribed by using the high capacity superscript III reverse transcriptase (Life Technologies) and the Mystic miRNA cDNA synthesis Mix kit (Sigma-Aldrich), respectively.
Techniques: Transfection, Labeling, Microscopy, Quantitation Assay, BrdU Incorporation Assay, Staining, Standard Deviation, BrdU Staining, Two Tailed Test