Bromodeoxyuridine (5-bromo-2 ..

Cell proliferation assay using 3H‐thymidine nucleotide incorporation and radioactive measurements.

Bromodeoxyuridine (BrdU) Antibodies and Kits

In addition to measuring relative rates of proliferation between different cells, cell proliferation assays can be useful for determining the effects of compounds that may have the potential for increasing or decreasing cell proliferation rates.

In a cell proliferation assay, ..

Besides overall metabolic activity, cell proliferation may be measured by examining one or more specific markers within a cell. A well-published example is the . In this assay, cells are treated with , a analog that is incorporated into the DNA during cell proliferation. The can be detected with a special antibody, and high rates of incorporation correlate with high cell proliferation rates. Another such marker is , which promotes DNA replication in actively proliferating cells.

To identify the potential acetylation sites on TopBP1, we performed in-silico analysis in combination with mass spectrometry analysis. We detected 7 lysine sites within TopBP1 that could be acetylated (K475, K482, K789, K825, K1253, K1398 and K1445) (Fig. B, C and : Figure S2). Because of a difficulty to predict which site is important for function of TopBP1, we performed site-directed mutagenesis to generate a TopBP1 expression construct containing three lysine residues mutated to arginine (TopBP1-3R: K475R, K789R and K825R) to mimic the deacetylated form. Comparison of the acetylation state of TopBP1 and TopBP1-3R revealed that these mutations exhibited a reduced acetylation level caused by SIRT1-HY over-expression (Fig. D). To further study the effect of these mutations on DNA replication, we transfected TopBP1-WT, TopBP1-3R (K475R, K789R and K825R), TopBP1-3Q (K475Q, K789Q and K825Q), TopBP1-5R (K475R, K789R, K825R, K1253R, and K1398R) and TopBP1-5Q (K475Q, K789Q, K825Q, K1253Q, and K1398Q), into SIRT1-WT and SIRT1-MT MEF cells and used them in a BrdU incorporation assay. The transfected cells were first treated with HU for 4 hours and then BrdU labeling was performed for 1 hour. As seen in Figure E, under all transfection conditions, SIRT1-MT cells exhibited a significantly higher percentage of BrdU+ cells than wild type cells, which is consistent with the view that SIRT1 regulates the activity of TopBP1 (Fig. E). In wild type MEF cells, over-expression of the deacetylated forms, TopBP1-3R and TopBP1-5R, significantly reduced the percentage of BrdU positive cells, whereas this effect was not observed when TopBP1-5Q was expressed. Instead, TopBP1-5Q significantly increased the fraction of BrdU incorporating cells in SIRT1-WT cells, mimicking the effect observed in SIRT1-MT MEF cells. Of note, there was minimal effect of these constructs in SIRT1-MT cells, except for TopBP1-5R, which significantly repressed BrdU incorporation. This result is consistent with the view that TopBP1-5R represents the highly activated form of TopBP1 that results from SIRT1 deacetylation.

Chemiluminescence‐based BrdU ELISA to measure DNA synthesis.

In an attempt to identify the role of NUAK1 inregulating the proliferation of MIA PaCa-2 cells, the cells weretransfected with NUAK1 plasmids. Following stable transfection,NUAK1 expression was detected by western blot analysis and theexpression of eight proliferation-associated markers of MIA PaCa-2cells (Ki67, PCNA, p27, p21, c-myc, CDK1, CDK2 and p53) was alsodetermined by western blot analysis. The results revealed thatNUAK1 plasmids evidently increased NUAK1 protein expression, andsuppressed p53, p21 and p27 expression and promoted CDK1, CDK2 andKi67 expression in the MIA PaCa-2 cells (). Moreover, the proliferationrates of the MIA PaCa-2 cells were examined by MTT assay. Theresults revealed that overexpression of NUAK1 significantlyincreased the proliferation rate of the MIA PaCa-2 cells and thatthis increase in cell proliferation occurred in a dose-dependentmanner (). This wasfurther revealed by BrdU incorporation assay, showing thattransfection with NUAK1 resulted in increased DNA synthesisactivity per viable cell in the MIA PaCa-2 cells also in adose-dependent manner ().

Thymidine and BrdU, Cell Proliferation Assay ..

MEF cells were cultured overnight in 10 mm dishes. The cells were then either directly labeled with BrdU (BD), or treated with 10 mM hydroxyurea (HU) for 4 hours and released into medium containing BrdU, or reverse transfected by LipofectamineTM 2000 (Invitrogen) and then followed with the indicated treatment. The detailed FACS analysis of BrdU-containing samples was performed as described [].

Bromodeoxyuridine (BrdU) | DNA/RNA Synthesis …

DNA synthesis analysis was assessed as described []. Briefly, cells were cultured in the logarithmic phase and labeled with 10 nCi of 14C-thymidine (NEN) for 24 hours in DMEM. The medium containing 14C was then removed and replaced with normal DMEM culture medium for another 24 hours. After the cells were irradiated and incubated accordingly, they were pulse labeled with 3H-thymidine (NEN) at 2.5 mCi/ml for 15 min. Cells were harvested, washed twice with PBS, and fixed overnight at -20C with 70% ethanol. The samples then were transferred onto Whatman filters and washed with 70% ethanol and 90% methanol sequentially. The dried filters were assayed with a liquid scintillation counter (Beckman, LS6000IC). The resulting ratio of 3H counts per minute to 14C counts per minute, corrected for the counts per minute that were the result of channel crossover, was a measure of DNA synthesis.