Journal: Marine drugs
Article Title: The Polybrominated Diphenyl Ether Bromoxib Disrupts Nuclear Import and Export by Affecting Nucleoporins of the Nuclear Pore Complex.
doi: 10.3390/md23030108
Figure Lengend Snippet: Figure 6. Bromoxib inhibits the expression of the short-lived anti-apoptotic Bcl-2 protein Mcl-1. (A) Schematic illustration of the effect of RNA polymerase II inhibition by the CDK9 inhibitor AZD4573 on the subsequent protein expression of short-lived proteins such as Mcl-1. RNA poly- merase II mediates the transcription of mRNA in the nucleus, which then translocates across the NPC into the cytosol. There, mRNA is translated at the ribosomes into respective proteins. Finally, proteins are degraded by the proteasome. One of the short-lived proteins is the anti-apoptotic Bcl-2 protein Mcl-1. Studies by Cidado et al. have shown that the selective CDK9-inhibitor AZD4573 inhibits the CDK9-mediated activation of RNA polymerase II and induces the downregulation of Mcl-1 mRNA, followed by a rapid downregulation of Mcl-1 at the protein level [25]. (B) Ramos cells were treated with 10 µM or 40 µM of bromoxib, or 0.1 µM of the CDK9 inhibitor AZD4573 in a kinetic for 0 h, 2 h, 4 h, 6 h or 8 h. Treatment with 0.2% w/v DMSO for 8 h served as diluent control. Subsequently, immunoblots for the anti-apoptotic Bcl-2 protein Mcl-1, the caspase substrate poly(ADP-ribose) polymerase-1 (PARP), or vinculin (loading control) were performed. Solid arrowheads indicate the uncleaved form of PARP (p116); open arrowheads indicate the cleaved form (p85). (C) Quantitative analysis of immunoblots (see (B) for representative immunoblots) from three independent biological replicates (n = 3). (D) To avoid caspase-mediated protein degradation, Ramos cells were pretreated with 10 µM of the caspase-inhibitor QVD for 30 min. Subsequently, cells were treated as described in (B). (E) Quantitative analysis of immunoblots (see (D) for representative immunoblots) from three independent biological replicates (n = 3).
Article Snippet: The pan-caspase inhibitor N-(2-Quinolyl)valylaspartyl-(2,6-difluorophenoxy)methyl ketone (Q-VD-OPh, QVD, #S7311) and the selective CDK9-inhibitor AZD4573 (S8719) were obtained from Selleckchem (Houston, TX, USA).
Techniques: Expressing, Inhibition, Activation Assay, Control, Western Blot
![Figure 6. Bromoxib inhibits the expression of the short-lived anti-apoptotic Bcl-2 protein Mcl-1. (A) Schematic illustration of the effect of RNA polymerase II inhibition by the CDK9 inhibitor <t>AZD4573</t> on the subsequent protein expression of short-lived proteins such as Mcl-1. RNA poly- merase II mediates the transcription of mRNA in the nucleus, which then translocates across the NPC into the cytosol. There, mRNA is translated at the ribosomes into respective proteins. Finally, proteins are degraded by the proteasome. One of the short-lived proteins is the anti-apoptotic Bcl-2 protein Mcl-1. Studies by Cidado et al. have shown that the selective CDK9-inhibitor AZD4573 inhibits the CDK9-mediated activation of RNA polymerase II and induces the downregulation of Mcl-1 mRNA, followed by a rapid downregulation of Mcl-1 at the protein level [25]. (B) Ramos cells were treated with 10 µM or 40 µM of bromoxib, or 0.1 µM of the CDK9 inhibitor AZD4573 in a kinetic for 0 h, 2 h, 4 h, 6 h or 8 h. Treatment with 0.2% w/v DMSO for 8 h served as diluent control. Subsequently, immunoblots for the anti-apoptotic Bcl-2 protein Mcl-1, the caspase substrate poly(ADP-ribose) polymerase-1 (PARP), or vinculin (loading control) were performed. Solid arrowheads indicate the uncleaved form of PARP (p116); open arrowheads indicate the cleaved form (p85). (C) Quantitative analysis of immunoblots (see (B) for representative immunoblots) from three independent biological replicates (n = 3). (D) To avoid caspase-mediated protein degradation, Ramos cells were pretreated with 10 µM of the caspase-inhibitor QVD for 30 min. Subsequently, cells were treated as described in (B). (E) Quantitative analysis of immunoblots (see (D) for representative immunoblots) from three independent biological replicates (n = 3).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_7294/pm40137294/pm40137294__page11_image1.jpg)