Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-3

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>�B/IκBα complexes in the nucleus after treatment with LMB or PMA. Ten micrograms of cytosolic and nuclear extracts from CDT cells treated with either PMA or LMB during 4 and 6 hours respectively were analyzed by Western Blot using antibodies against p65/RelA and IκBα. Immunoprecipitation assays were performed using 100 μg of these cytosolic and nuclear extracts, which were incubated with 5 μg of an antibody against p65/RelA conjugated with agarose. IκBα and p65/RelA complexes were characterized by immunoblotting. (b) Contamination with cytosolic proteins during nuclear protein extraction or accumulation of cytosolic proteins in the nucleus after treatment with LMB was assessed by Western Blot using an antibody against both p105 and p50/NF-κB1 proteins. (c) Analysis of NF-κB DNA-binding activity in CDT cells treated with either PMA or LMB. Three micrograms of nuclear extract were incubated with an oligonucleotide containing the double consensus motif κB present in the HIV LTR labeled with [α-P]-dCTP. Protein extracts were obtained from CDT cells after treatment with either LMB or PMA for 6 and 4 hours respectively. (d) Analysis of IκBα pool dependence on protein synthesis. Ten micrograms of nuclear extracts from CDT cells incubated with 20 nM LMB for 4 hours and 10 μg/ml CHX and/or 25 ng/ml PMA for 4 hours,30 min and 2 hours, respectively, were analyzed by Western Blot

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-0

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>antibodies against IκBα and p65/RelA. A secondary antibody conjugated with Texas Red (Molecular Probes) was used. Images were taken by confocal microscopy

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-4

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>expression vectors or (b) pcDNA3.1 and/or CMV-Tat and/or CMV-IκBα expression vectors, as indicated. Cells were treated with LMB immediately after transfection and/or with PMA two hours after transfection, as indicated. Luciferase activity was measured 18 hours after transfection. Numbers on the top of the bars represent fold transcriptional activity relative to unstimulated T cells transfected with pcDNA3.1

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-8

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-7

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>ctors per million of cells. LMB was added immediately after transfection. After 18–24 hours of incubation, cells were analyzed by confocal microscopy. pEYFP-C1 vector was used as control of unspecific distribution. (b) Resting purified CDT lymphocytes were transiently transfected with the control plasmid pcDNA3.1 by using an Amaxa nucleofector and a classical electroporator (Equibio). As occurs in untransfected resting T cells (lane 1), NF-κB was not induced in resting CDT lymphocytes after electroporation (lanes 3 and 4). As a positive control, NF-κB (p50/p65) binding was induced in these cells by PMA activation (lane 2)

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-1

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>ctors per million of cells. LMB was added immediately after transfection. After 18–24 hours of incubation, cells were analyzed by confocal microscopy. pEYFP-C1 vector was used as control of unspecific distribution. (b) Resting purified CDT lymphocytes were transiently transfected with the control plasmid pcDNA3.1 by using an Amaxa nucleofector and a classical electroporator (Equibio). As occurs in untransfected resting T cells (lane 1), NF-κB was not induced in resting CDT lymphocytes after electroporation (lanes 3 and 4). As a positive control, NF-κB (p50/p65) binding was induced in these cells by PMA activation (lane 2)

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-2

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-5

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>together with CMV-IκBα or pcDNA3.1 as negative control, and then activated with anti-CDand IL-2, PHA and IL-2, or maintained in the absence of activation. Viral replication was determined by quantification of HIV p24-gag antigen in culture supernatants (a) after 5 days of transfection or (b) after 7 days of transfection. Numbers on the top of the bars represent fold HIV-replication relative to unstimulated T cells transfected with pcDNA3.1. Differences in p24-gag production were significant for resting and anti-CD-activated T cells (p < 0.05) and a trend towards statistical significance was found in PHA-activated T cells (p = 0.081)

Treatment of Jurkat-Tat101 cells with doxycycline reduced the expression of hsa-miR-21, -222, -29a, and -1290.

<p>(A) Jurkat-Tat101 cells were co-transfected with pLTR-LUC and pEGFP, as control of transfection efficiency. Cells were treated or not with doxycycline 1μg/ml immediately after transfection. After incubation for 18 hours, the expression of luciferase (left graph) and EGFP (right graph) was analyzed by chemiluminescence and flow cytometry, respectively. Data shown are media and SEM from three independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (**** for <i>p</i><0.0001). (B) Expression of hsa-miR-21, -222, -29a, and -1290 was analyzed by qRT-PCR in Jurkat-Tat101 cells treated or not with doxycycline for 18 hours. Media and SEM from three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (**** for <i>p</i><0.0001).</p

Analysis of PTEN-AKT-FOXO3a signaling pathway in Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 cells.

<p>Expression of hsa-miR-21 and hsa-miR-222 was measured in Jurkat-hsa-miR-21 (A) and Jurkat-hsa-miR-222 (B), respectively, by qRT-PCR. (C) The expression of PTEN, pAKT^S473 and total AKT was analyzed by immunoblotting in cytosolic protein extracts from Jurkat-hsa-miR-21, Jurkat-hsa-miR-222 and Jurkat-hsa-miR-Null as control cells. (D) Analysis of the rate of apoptosis in Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 in comparison with Jurkat-hsa-miR-Null control cells. Apoptosis was measured by chemiluminescence after treatment with FasL for 4 hours. Media and SEM from at least three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (*, ** or *** for <i>p</i><0.05, <i>p</i><0.01 or <i>p</i><0.001, respectively).</p