Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways-0

D with 1 μM BMS-345541. The IKKβ activities were examined by kinase assay using GST-IκBα as a substrate. The [γ-P]-labeled IκB-α protein was visualized by autoradiography. The IKKβ activities were quantitated by ImageQuant software. The bottom panel shows a commassie blue staining of GST-IκBα to show equal amount of substrate in each reaction. BMS-345541 inhibited IKKβ activity in C8166 cells in dose-dependent manner; however, Purvalanol A had no effect on IKKβ. Kinase assay were performed as described above using 0.01, 0.1, and 1 μM of BMS-345541 and 1, 10 μM of Purvalanol A. The stained gel below is a representative of the kinase reaction.<p><b>Copyright information:</b></p><p>Taken from "Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways"</p><p>http://www.aidsrestherapy.com/content/5/1/12</p><p>AIDS Research and Therapy 2008;5():12-12.</p><p>Published online 10 Jun 2008</p><p>PMCID:PMC2483717.</p><p></p

Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways-2

Rn blot analysis using anti-IκB, phospho IκB (ser 32), p65, phospho p65 (ser 536), p50, p52, Tax and actin. Twenty five microgram of each extract was used to separate on a 4–20% SDS/PAGE. Levels of total IκB and p65 did not change between cell types, however there was a dramatic increase of phosphor-IκB and phosphor-p65 in HTLV-1 infected cells and their suppression by BMS-345541 which inhibits IKKβ activity in vivo.<p><b>Copyright information:</b></p><p>Taken from "Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways"</p><p>http://www.aidsrestherapy.com/content/5/1/12</p><p>AIDS Research and Therapy 2008;5():12-12.</p><p>Published online 10 Jun 2008</p><p>PMCID:PMC2483717.</p><p></p

Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways-4

Ned with a mixture of propidium iodide buffer followed by cell sorting analysis. The acquired FACS data were analyzed by ModFit LT software (Verity Software House, Inc.).<p><b>Copyright information:</b></p><p>Taken from "Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways"</p><p>http://www.aidsrestherapy.com/content/5/1/12</p><p>AIDS Research and Therapy 2008;5():12-12.</p><p>Published online 10 Jun 2008</p><p>PMCID:PMC2483717.</p><p></p

Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways-1

Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown). Detection of apoptosis through annexin V and PI staining. Cells were washed three times in PBS and re-suspended in binding buffer, stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer.<p><b>Copyright information:</b></p><p>Taken from "Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways"</p><p>http://www.aidsrestherapy.com/content/5/1/12</p><p>AIDS Research and Therapy 2008;5():12-12.</p><p>Published online 10 Jun 2008</p><p>PMCID:PMC2483717.</p><p></p

Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR-2

H "no substrate" and histone H3 N-terminal mutant (K to A at positions 4, 9, 14, 18, 23, 27, 36, and 37) serve as negative controls. Wild type Tat 1–86 protein was used for methylation assay. The panel shows the incorporation of -H onto the Tat mutant peptides. Tat K50A showed a ~2 fold drop in counts, whereas the K51A showed more than ~10 fold drop in activity. Purified biotin labeled TAR RNA or PolyU RNA was mixed with purified proteins including wild type Tat 1–86, Tat 101, methylated Tat 101, purified Cdk9/cyclin T (data not shown) or extract. Unmodified and methylated Tat (1–86 and 1–101) were incubated with CEM nuclear extract containing endogenous Cdk9/cyclin T complexes (both active and inactive small and large complexes). Biotin-TAR RNA was added to the reaction mixture at the same time, processed and western blotted for presence of cyclin T.<p><b>Copyright information:</b></p><p>Taken from "Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR"</p><p>http://www.retrovirology.com/content/5/1/40</p><p>Retrovirology 2008;5():40-40.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2412914.</p><p></p

Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR-1

The negative control; Lanes 2–4 titration of Tat from 0.01, 0.1 and 1.0 ug to establish a range of activation; Lanes 5–10 are in the presence of 0.1 ug Tat as well as the indicated transfected siRNAs. TZM-bl cells containing an integrated LTR-Luc were transfected with siGFP, siSETDB1, siTIF-1, siG9a, and siHP1 in addition to Tat (0.1 ug) to initiate transcription. Confirmation of the knockdown of SETDB1 is shown in a Western blot below. Each transfection and luciferase assay was repeated at least three times.<p><b>Copyright information:</b></p><p>Taken from "Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR"</p><p>http://www.retrovirology.com/content/5/1/40</p><p>Retrovirology 2008;5():40-40.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2412914.</p><p></p

Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR-0

4) peptide immunoprecipitated complexes were probed for the presence of SETDB1, SETDB2, and SUV39H1. 1/20 of input was used as positive control for western blots. Biotin-labeled wild type Tat (Lane 2) and acetylated Tat (Lane 3) peptide complexes were probed for the presence of bound G9a and SETMAR. Positive control reaction using BRG1 pulldown for the acetylated Tat [61], and competition experiment with Tat 42–51 peptide (1:10 ratio) as well as purified wild type Tat 1–86 (1:10 ratio) to compete out SETDB1 binding. GST-bound wild type Tat and wild type Tax protein complexes were probed for the presence of bound SETDB1 and SETDB2. A summary of the Tat binding interactions between all members of the SUV39 family as predicted by SMART) [73]. Under both the Unmodified Tat and Acetylated Tat binding affinity column, a "-" indicates that the enzyme does not bind to the indicated form of Tat, while increasing amounts of "+" indicates that the enzyme bound to the indicated form of Tat with a greater specificity. The "UN" indicates that binding affinities were undetermined.<p><b>Copyright information:</b></p><p>Taken from "Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR"</p><p>http://www.retrovirology.com/content/5/1/40</p><p>Retrovirology 2008;5():40-40.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2412914.</p><p></p

Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR-3

Were subsequently removed and incubated with Tat for 4 hrs at 37°C in RPMI without serum. Cells were then plated in complete media for 6 days at 37°C and supernatants were process for RT activity. The effect of purified Tat protein on HLM-1 activation (lane 2) and subsequent super-activation with siSETDB1 and Tat protein in HLM-1 cells (lane 3). Lane 4 was with siHP1 and lane 5 with siCDK scrambled RNA. Western blot of transfected cells for SETDB1, HP1 and actin. Cell extracts were processed post siRNA transfection and western blotted for various proteins. For the actin westerns, Lane 1 is from siSETDB1 treatment and lane 2 is from siHP1 treatment.<p><b>Copyright information:</b></p><p>Taken from "Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR"</p><p>http://www.retrovirology.com/content/5/1/40</p><p>Retrovirology 2008;5():40-40.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2412914.</p><p></p