Article Figures & Data
Figures
- Fig. 1.
Survivin expression in irradiated human umbilical vein endothelial cells (HUVECs) and various cancer cells. Cells were irradiated with 3 Gy. Total cellular protein was extracted at the indicated time points after treatment. Shown are the Western immunoblots using antibodies against survivin and α-tubulin. A, HUVECs; B, cancer cell lines; C, G2-M percentage of HUVEC cells at 0, 4, and 24 h after irradiation Each bar, the mean + SD based on three separate experiments.
- Fig. 2.
Down-regulation of survivin mRNA by 3 Gy in human umbilical vein endothelial cells (HUVECs), but not in colorectal cancer cells. Cells were irradiated with 3 Gy. Total RNA was extracted at the indicated time points after the irradiation treatment. The RNA samples were reverse-transcribed to cDNA and were quantified by Taqman real-time PCR. Shown are the relative mRNA levels of survivin in reference to β-actin. A, HUVECs; B, SW480 and SW620
- Fig. 3.
Transcriptional down-regulation of survivin after irradiation. Human umbilical vein endothelial cells (HUVECs) were treated with actinomycin D and were irradiated 1 h later. Total RNA was extracted at the indicated time points after the irradiation. The RNA samples were reverse-transcribed to cDNA and were quantified by Taqman real-time PCR. Shown are the relative mRNA levels of survivin (A) or Cox-2 (B) in reference to β-actin. C, HUVEC cells were transiently transfected with the SP1 plasmid. Transfected cells were irradiated and assayed at the various time points.
- Fig. 4.
Role of p53 in transcriptional down-regulation of survivin by irradiation: human umbilical vein endothelial cells (HUVECs) were irradiated with 3 Gy. Total cellular protein was extracted at the indicated time points after treatment. Shown are the Western immunoblots (A) using antibodies against total p53 or phospho-p53. B, the relative mRNA levels of survivin in reference to β-actin in HUVECs after transduction of Ad.LacZ or Ad.p53. C, the relative mRNA levels of survivin in reference to β-actin in irradiated Val138 cells cultured at either 32 degrees or 39 degrees D, the promoter activity of survivin in HUVECs cotransfected with the SP1 plasmid plus either a control plasmid or an expression plasmid of mutant p53.
- Fig. 5.
Survivin affects growth and apoptosis in response to irradiation. A, 293 cell lines stably transfected with or without the survivin-overexpressing plasmid were irradiated with 3 Gy. Total cellular protein was extracted at the indicated time points after the treatment. Shown are the Western immunoblots using an antibody against cleaved caspase 3, 293-neo, 293 cells stably transfected with pCDNAhis-neo; 293-survivin, 293 cells stably transfected with pCDNAhis-survivin. B, 293 cell lines with or without survivin-expressing plasmid were irradiated with 3 Gy. Cell viability was determined by the 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Each column, the mean + SD from three repeated experiments.
- Fig. 6.
Inhibition of survivin enhanced radiation effects in H460 lung cancer cells. A, survivin expression was attenuated 48 h after H460 cells were transfected with anti-survivin antisense oligonucleotide (AS). Mismatch (MS) is the control oligonucleotide. B, H460 cells were transfected with either AS or MS oligonucleotides. 24 h later, the transfected cells were treated with or without 3 Gy. The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed 48 h after irradiation. Each graph, the mean + SD from three repeated experiments. C, transfected H460 cells or nontransfected control were treated with 0–5 Gy. After 2 weeks, colonies were stained and colonies over 50 cells were scored. Data are shown as the mean ± SD.
Tables
- Table 1
Primer and probe sequences for reverse transcription PCR
Primer and probe sequences Survivin Forward primer 5′-TGC CCC GAC GTT GCC-3′ Reverse primer 5′-CAG TTC TTG AAT GTA GAG ATG CGG T-3′ Probe 6FAM-5′-CCT GGC AGC CCT TTC TCA AGG ACC-3′-TAMRA β-actin Forward primer 5′-TGAGCGCGGCTACAGCTT-3′ Reverse primer 5′-TCCTTAATGTCACGCACGATTT-3′ Probe 6FAM-5′-ACCACCACGGCCGAGCGG-3′-TAMRA COX-2 Forward primer 5′-GCTCAAACATGATGTTTGCATTC-3′ Reverse primer 5′-GCTGGCCCTCGCTTATGA-3′ Probe 6FAM-5′-TGCCCAGCACTTCACGCATCAGTT-3′-TAMRA
Volume 64, Issue 8
