Figure 3.
Expression levels and activity of proteins involved in cell cycle, signaling, and apoptosis in response to treatment with celecoxib or DMC. RPMI8226 and 8226/Dox40 cells were cultured in the presence of 30 and 50 μM celecoxib (CXB) or 20 and 40 μM DMC for 48 hours as indicated. In parallel, some cell cultures received 0.5 μM doxorubicin (Dox). Total cellular lysates were prepared and analyzed by Western blotting with specific antibodies or by in vitro kinase assay. (A) Analysis of the cell-cycle regulatory proteins cyclin A (cycA) and cyclin B (cycB), and the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. \({\otimes}\)
indicates that RPMI8226 cells were treated with doxorubicin as well; however, due to efficient killing of these drug-sensitive cells, the recovered amount of protein was insufficient for analysis. (B) Analysis of the activity of representatives of 3 major signaling pathways, the mitogenic MAP kinase pathway, the Akt/PKB survival pathway, and the antiapoptotic NF-κB pathway. The activity of MAP kinase kinases 1 and 2 (MEK1 and MEK2) is determined with the use of antibodies that specifically recognize the phosphorylated (ie, active) forms of MEK1 and MEK2 (p-MEK1/2). The activity of Akt is determined with the use of antibodies that recognize Akt phosphorylated on either threonine-308 (p-Akt, thr-308) or on serine-473 (p-Akt, ser-473). In comparison, the total amount of Akt (active and inactive) is shown in the panel labeled Akt. The activity of NF-κB signaling is indicated by in vitro kinase assays measuring the activity of IκB kinase (IKK), which acts as an activator of NF-κB via the phosphorylation (and thus inactivation) of the NF-κB inhibitor, IκB. The panel labeled 32P-IκBα is an autoradiograph reflective of IKK enzymatic activity, whereas the panel labeled IκBα shows Coomassie blue staining of the same gel to confirm that equal amounts of substrate (IκBα) were used in each reaction. (C) Analysis of proteins involved in apoptosis. Activation of caspase-3 is revealed via the detection of the active caspase p17 fragment, and via the emergence of cleaved PARP, which is a substrate of caspase-3. Also shown is survivin, an antiapoptotic protein that is often found elevated in tumor cells.
![Figure 3. Expression levels and activity of proteins involved in cell cycle, signaling, and apoptosis in response to treatment with celecoxib or DMC. RPMI8226 and 8226/Dox40 cells were cultured in the presence of 30 and 50 μM celecoxib (CXB) or 20 and 40 μM DMC for 48 hours as indicated. In parallel, some cell cultures received 0.5 μM doxorubicin (Dox). Total cellular lysates were prepared and analyzed by Western blotting with specific antibodies or by in vitro kinase assay. (A) Analysis of the cell-cycle regulatory proteins cyclin A (cycA) and cyclin B (cycB), and the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\otimes}\) \end{document} indicates that RPMI8226 cells were treated with doxorubicin as well; however, due to efficient killing of these drug-sensitive cells, the recovered amount of protein was insufficient for analysis. (B) Analysis of the activity of representatives of 3 major signaling pathways, the mitogenic MAP kinase pathway, the Akt/PKB survival pathway, and the antiapoptotic NF-κB pathway. The activity of MAP kinase kinases 1 and 2 (MEK1 and MEK2) is determined with the use of antibodies that specifically recognize the phosphorylated (ie, active) forms of MEK1 and MEK2 (p-MEK1/2). The activity of Akt is determined with the use of antibodies that recognize Akt phosphorylated on either threonine-308 (p-Akt, thr-308) or on serine-473 (p-Akt, ser-473). In comparison, the total amount of Akt (active and inactive) is shown in the panel labeled Akt. The activity of NF-κB signaling is indicated by in vitro kinase assays measuring the activity of IκB kinase (IKK), which acts as an activator of NF-κB via the phosphorylation (and thus inactivation) of the NF-κB inhibitor, IκB. The panel labeled 32P-IκBα is an autoradiograph reflective of IKK enzymatic activity, whereas the panel labeled IκBα shows Coomassie blue staining of the same gel to confirm that equal amounts of substrate (IκBα) were used in each reaction. (C) Analysis of proteins involved in apoptosis. Activation of caspase-3 is revealed via the detection of the active caspase p17 fragment, and via the emergence of cleaved PARP, which is a substrate of caspase-3. Also shown is survivin, an antiapoptotic protein that is often found elevated in tumor cells.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/106/13/10.1182_blood-2005-07-2819/2/zh80240588300003.jpeg?Expires=1724286183&Signature=DDPofQ4MG6m9kBu2WOzAMfHKRFA~RlZ41q-GNKjUnaCDiVO6yUwIdrdooV3dp-ZwIgxcTV5dKfoKFccFCTpIdcBo1GdwLbSd7cmcZLoWh7CaWNKVJH85uPPq7nP438HOmrr5UkZD1KEZOua9GlAJzZllThIJEZFEP4rU8Gvw5kt3z8Aos4GrjpranGRY7FRmwnhjz4SBx2AmnVJhEGbVxAUPeM58vuW0GVWxCNIUDn9xojXeev6ruLVELOuG1N3XePsUrT37zwrVpNM6faAvG83Xsvjd-2BewgFhe9Av5z4aEgpsS7TyvyoZthSkOkCIzcG4nL4Orke8Ninfa6~Byg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
