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

Ro5-3335 RUNX inhibitor synergizes with SAHA HDAC inhibitor.

<p><b>A</b>) JLat cells were cultured in the presence of increasing concentrations of Ro5-3335 in the presence or absence of 1 μM SAHA. Seventy-two hours after treatment the percentage of GFP positive cells was determined by flow cytometry. <b>B</b>) ACH2 cells were cultured with Ro5-3335 and 10 μM SAHA as in panel A. Forty-eight hours post treatment RNA was extracted from the cells and used to determine the relative increase in HIV-1 Gag mRNA as normalized to GAPDH. <b>C</b>) TZMbl cells were cultured with Ro5-3335 and 10 μM SAHA as above. Forty-eight hours post treatment RNA was extracted from the cells and used to determine the relative increase in HIV-1 U5 mRNA as normalized to GAPDH. <b>D</b>) Jlat, <b>E</b>) ACH2 and <b>F</b>) TZMbl were cultured in the presence of the indicated concentrations of SAHA. Forty-eight hours post treatment RNA was extracted from the cells and used to determine the relative changes in RUNX1, CBF-β or RUNX3. <b>G</b>) ACH2 or <b>H</b>) TZMbl cells were transfected with 50 pMol siRNA against RUNX1 and/or treated with 10 μM SAHA at twenty-four hours post transfection. Forty-eight hours post transfection RNA was extracted and expression measured as above. * p≤0.05, ** p≤0.01 and *** p≤0.001</p

RUNX1 expression in memory CD4+ T-cells correlates with clinical metrics in patients.

<p>CD4+ memory T-cells were sorted from total PBMC of viremic HIV-1 patients in the absence of therapy. RNA extract from these cells was used to measure the expression of promoter proximal and distal RUNX1 as normalized to GAPDH. Expression level were then graphed as follows: <b>A</b>) promoter proximal RUNX1 vs viral load, <b>B</b>) promoter proximal RUNX1 versus CD4+ T-cell count, <b>C</b>) promoter distal RUNX1 versus viral load and <b>D</b>) promoter distal RUNX1 versus CD4+ T-cell count. R and P values were computed using Spearman test.</p

Ro5-3335 improves re-activation of HIV-1 in patient samples.

<p>PBMCs from HIV-1 patients suppressed on therapy were treated with DMSO, 250 nM SAHA, 5 uM Ro5-3335 or both drugs in combination. PMA treatment was used as a positive control. <b>A</b>) Twenty-four hours after treatment RNA was isolated from cells and used to detect Gag mRNA by RT-qPCR. <b>B</b>) Flow cytometry was used to determine the percentage of activated T-cells by staining for intracellular Ki67.</p

HIV-1 Vif sequesters CBF-β in the cytoplasm.

<p><b>A</b>) HeLa CBF-β knockdown (KD) cells (5×10⁶) were cotransfected with the Vif expression vector pNL-A1 (2.5 μg) and/or the CBF-β vector pCBF-β (1 μg). Total amounts of transfected DNA were adjusted to 5 μg using empty vector DNA as appropriate. Three hours after transfection, cells were trypsinized and seeded onto cover slips. Cells were fixed 24 hr later in methanol (10 min, -20°C) and then stained with rabbit antibodies to Vif (Vif93; 1:100) or CBF-β (Thermo Fisher; 1:100). Bound antibodies were visualized by Texas-Red or Cy2-conjugated secondary antibodies (Jackson Labs; 1:100). Images were collected on a Zeiss LSM410 confocal microscope using a Plan-Apochromat 63x/1.4 oil immersion objective (Zeiss). <b>B</b>) 293T cells were seeded in a 6-well plate and transfected with 0.1 ug pLTR-GL3 and 0.04, 0.2 and 1 ug pVif or pVif ΔD. Forty eight hours post transfection cells were collected and used to prepare protein extracts. Cell extracts were then used to determine the production of luciferase. Results are displayed as luciferase signal relative to transfection of reporter construct alone. <b>C</b>) 293T cells were seeded in a 6-well plate and transfected with 0.1 ug pLTR-GL3 mutant 2 and 1 ug of pVif or pVif ΔD. Luciferase activity was determined as above. Jurkat cells were transfected with GFP plasmid and <b>D</b>) RUNX1 or <b>E</b>) CBF-β plasmid and infected twenty-four post transfection with equivalent doses of NL4-3 WT or NL4-3 ΔVif. RT values were determined at days 2, 4, 6, 8 and 10 post infection. Data is presented as RT activity relative to control (GFP transfected) cells. ** p≤0.01 and *** p≤0.001</p

RUNX1 and CBF-β are capable of repressing HIV-1 replication.

<p><b>A</b>) HeLa cells were transfected with the pNL4-3 proviral plasmid and the indicated amounts of HA-tagged RUNX1 or CBF-β expression vectors. Forty-eight hours post transfection cells were harvested, protein extracts were prepared and extracts were Western blotted for p24 and p55 Gag using human immune serum (upper panel), β-actin (middle panel) or HA (lower panel). Jurkat T-cells were transfected with1 ug of pMAXGFP or 0.04, 0.2 or 1 ug of <b>B</b>) pRUNX1 or <b>C</b>) pCBF-β and infected with NL4-3 twenty-four hours post transfection. Viral replication was followed by RT at days 2, 4, 6, 8 and 10 post infection.</p

RUNX1 and CBF-β suppress LTR-driven promoter expression of the integrated and unintegrated LTR.

<p><b>A</b>) TZMbl indicator cells were seeded in a six well plate and transfected with 0.01 μg Tat expression vector and 1 μg of RUNX1 or CBF-β expression vectors or a combination of the two. Forty-eight hours after transfection cells were lysed and β-galactosidase activity was measured by a luminescence assay. β-galactosidase is represented as relative to transfection of Tat alone. <b>B</b>) 293T cells were seeded in a 6-well plate and transfected with pLTR-GL3 and RUNX1 or CBF-β vectors as indicated. Forty-eight hours after transfection the cells were harvested and cell lysates prepared. Cell extracts were then used to determine the production of luciferase. Results are displayed as luciferase signal relative to transfection of reporter construct alone. Western blotting was performed on transfected cells to assess the levels of RUNX1 and CBF-β expression. * p≤0.05, ** p≤0.01 and *** p≤0.001</p