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Transfection with pU, pM and pUM in combination with radiation specifically down regulates expression of uPAR and MMP-9 and induces apoptosis.

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posted on 12.09.2012, 02:18 by Ramaprasada Rao Kotipatruni, Arun Kumar Nalla, Swapna Asuthkar, Christopher S. Gondi, Dzung H. Dinh, Jasti S. Rao

Daoy and D283 cell lines were transfected with either transfection reagent alone (control, Con), pSV (scrambled vector) or gene specific shRNA’s as described in materials and methods. A) RT-PCR analysis and western blotting was carried to determine the expression levels of uPAR and MMP-9 in cell transfected with reagent, pSV, pU-, pM- and pUM-transfected Daoy and D283 cells (with and without radiation, 8 Gy). The experiments were repeated three times and representative images were shown. The immunoblots were stripped and re-probed with GAPDH as a loading control. Semi-quantitative RT-PCR analysis was carried out to detect mRNA levels of uPAR and MMP-9 using specific primers. B) Protein band and PCR amplicon intensities were quantified by densitometry analysis using ImageJ software (National Institutes of Health). The levels of uPAR and MMP-9 protein were normalized to GAPDH levels in mock-transfected cells. Columns: mean of triplicate experiments; bars: s.d.; *p<0.01, significant difference from pSV-transfected cells. C) 72 hrs after Transfection (with or without radiation), the cells were trypsinized and analyzed by flow cytometry to measure the number of apoptotic TUNEL-positive cells using the APO-BrdU TUNEL Assay kit. The percent apoptotic cells from each treatment are represented and the mean ± s.d. from three separate experiments was represented; *p<0.05 and ** p<0.01 were considered significant compared to pSV-transfected cells and pSV+IR treated cells, respectively. D) Apoptotic cells were also quantified using an Annexin V assay followed by FACS analysis. Results are reported as the percent of cells (minimum 10,000 analyzed) that were Annexin V-positive. Gating was based on positive and negative control cells. Representative FACS data of Daoy and D283 are shown (n = 3).

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