![Figure 2. Genomic instability leads to premature senescence in Tax-expressing cells. (A) Tax-transduced PBMCs during either early (< 1 month) or late (> 6 months) passage were fixed, stained with DAPI (4,6 diamidino-2-phenylindole), and then analyzed for the presence of cell division abnormalities such as asymmetric division, multinucleated cell, micronuclei, and anaphase bridges. (B) Karyotype analysis of Tax-transduced PBMCs during late passage. Arrows indicate telomere associations (Tas). (C) Western blot of Tax expression in early and late Tax-transduced PBMCs (Tax/PBMC no. 3). (D) Real-time PCR analysis of hTERT expression between early and late Tax-transduced PBMCs (Tax/PBMC no. 3). Normalized values are represented as the percentage of activation of early Tax-transduced PBMCs. (E) TRAP analysis of telomerase activity between early and late Tax-transduced PBMCs (Tax/PBMC no. 3). 3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS) buffer was a negative control. (F) Fluorescence in situ hybridization (FISH) of telomere sequences in early and late passage Tax-transduced PBMCs (Tax/PBMC no. 3) compared with MT2 cells. Histograms represent the intensities of fluorescence spots of telomere FISH staining with their respective numbers in each cell line. A minimum of 100 spots was quantified for each cell line. (G) Double immunofluorescence staining of late-cultured Tax-transduced PBMCs (Tax/PBMC no. 3) with anti-TRF2 and anti–phospho-H2AX antibodies. The contact of telomere-induced foci (red) and DNA double-strand breaks (green) is indicated by the arrows in the overlay image. (H) Immunofluorescence of p53 transcriptional activity in Tax-transduced PBMCs (PBMC/Tax no. 3) as indicated by phosphorylation on serine 20 of p53. (I) Real-time PCR expression of p53-responsive genes p21WAF and mdm2 in nontransduced PBMCs, and early and late passage Tax-transduced PBMCs (Tax/PBMC no. 3). Assays were performed in duplicate. Fold changes were calculated by comparing values with that of nontransduced PBMC normalized gene expression. Error bars represent the CT SD of the gene of interest.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/12/10.1182_blood-2009-08-241117/5/zh89991050090002.jpeg?Expires=1724239561&Signature=FnNPK5gPUT8Rm7Kyz9HMbFNvUlPi5Np~ZKn3-iPR9eQQVbAk5KLJ8-Oqde63j4yxR600MbZHe8p6P1NfP9xC4VV6~uSIU2csIi-TC2UVaj4PfdDXkyc6l6TtszD4FlwvPBfwfvFdAlMR~DjSFyNKfbJ1nycVJNJqWdW3Cn1MWg~JT6yl~naOQu6pugHsBmodTcwyfNXYAuB45fSH9o0Nny8kcNd9fhXKoXsfCQrrBI0xojX0TMehZBjDpvRhJaRNTykFH3kpnVFzoK~iFDgcdUEBUl07NKpvFTO6H7FhMC~bEvUkBUCY237OX6xBHIBqfvd6rmqxV5jkMOD5QwsyRw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Genomic instability leads to premature senescence in Tax-expressing cells. (A) Tax-transduced PBMCs during either early (< 1 month) or late (> 6 months) passage were fixed, stained with DAPI (4,6 diamidino-2-phenylindole), and then analyzed for the presence of cell division abnormalities such as asymmetric division, multinucleated cell, micronuclei, and anaphase bridges. (B) Karyotype analysis of Tax-transduced PBMCs during late passage. Arrows indicate telomere associations (Tas). (C) Western blot of Tax expression in early and late Tax-transduced PBMCs (Tax/PBMC no. 3). (D) Real-time PCR analysis of hTERT expression between early and late Tax-transduced PBMCs (Tax/PBMC no. 3). Normalized values are represented as the percentage of activation of early Tax-transduced PBMCs. (E) TRAP analysis of telomerase activity between early and late Tax-transduced PBMCs (Tax/PBMC no. 3). 3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS) buffer was a negative control. (F) Fluorescence in situ hybridization (FISH) of telomere sequences in early and late passage Tax-transduced PBMCs (Tax/PBMC no. 3) compared with MT2 cells. Histograms represent the intensities of fluorescence spots of telomere FISH staining with their respective numbers in each cell line. A minimum of 100 spots was quantified for each cell line. (G) Double immunofluorescence staining of late-cultured Tax-transduced PBMCs (Tax/PBMC no. 3) with anti-TRF2 and anti–phospho-H2AX antibodies. The contact of telomere-induced foci (red) and DNA double-strand breaks (green) is indicated by the arrows in the overlay image. (H) Immunofluorescence of p53 transcriptional activity in Tax-transduced PBMCs (PBMC/Tax no. 3) as indicated by phosphorylation on serine 20 of p53. (I) Real-time PCR expression of p53-responsive genes p21WAF and mdm2 in nontransduced PBMCs, and early and late passage Tax-transduced PBMCs (Tax/PBMC no. 3). Assays were performed in duplicate. Fold changes were calculated by comparing values with that of nontransduced PBMC normalized gene expression. Error bars represent the CT SD of the gene of interest.
