13 research outputs found
Model of ATM-KAP1-mediated regulation of EBV replication.
<p>i. KAP1 represses EBV lytic genes in latently infected cells. ii. Lytic cycle activation signals cause ATM-mediated phosphorylation of KAP1 at S824; phosphorylated KAP1 is unable to repress EBV lytic genes. These events allow expression of lytic genes of all kinetic classes in response to lytic cycle activation triggers. Events triggering the initial expression of ZEBRA and activation of ATM are unclear and shown by dashed arrows. Chloroquine, known to activate ATM, causes phosphorylation of KAP1 at S824 resulting in activation of EBV lytic cycle; IE: immediate early, E: early, L: late lytic genes.</p
Chloroquine induces phosphorylation of KAP1 at S824 and activates EBV lytic cycle in Burkitt lymphoma cells.
<p><b>A.</b> HH514-16 BL cells were treated with chloroquine (CQ) or chloroquine plus KU-55933 (CQ+KU) and harvested at 24 hours followed by staining with anti-phospho KAP1 (S824) plus anti-ZEBRA antibodies and visualized at 1000X magnification. <b>B.</b> HH514-16 BL cells were treated with chloroquine and harvested at different times post-treatment for immunoblotting with antibodies as indicated. <b>C.</b> HH514-16 cells were transfected with pFLAG-CMV2-KAP1 (wt) or pFLAG-CMV2-KAP1-S824A. After 48 hours, cells were treated with chloroquine or left untreated for another 48 hours and harvested for immunoblotting with indicated as antibodies. Numbers below bands indicate relative amounts of ZEBRA after normalization to β-actin. <b>D.</b> HH514-16 cells were treated with chloroquine (black bars) or left untreated (open bars), harvested 24 hours after treatment and relative levels of transcripts from EBV lytic genes <i>BZLF1</i>, <i>BMRF1</i> and <i>BFRF3</i> were determined by qRT-PCR after normalization to 18S rRNA using the ΔΔC<sub>T</sub> method. <b>E.</b> HH514-16 cells were untreated (open bar), treated with chloroquine (CQ; black bar: 10μM; horizontal striped bar: 200μM) or NaB (vertical striped bar), released virus particles were pelleted from supernatant 7 days later, treated with DNase, and quantified using q-PCR; ND: not detectable. <b>F.</b> BL cell lines Jijoye and Raji were treated with chloroquine (CQ) and harvested at different times post-treatment for immunoblotting with antibodies as indicated. Error bars for D and E represent 3 technical replicates from 2 experiments.</p
ATM induces phosphorylation of KAP1 at S824 upon exposure to lytic trigger.
<p><b>A.</b> HH514-16 BL cells were treated with increasing amounts of PI3 kinase-related kinase inhibitors (KU-55933 or Torin1; left panels) or PI3KK inhibitors plus NaB (right panels). After 24 hours, cells were harvested and lysates analyzed via immunoblotting with the antibodies indicated. <b>B.</b> HH514-16 cells were treated with NaB (upper panel) or NaB plus KU-55933 (1μM; middle panel) or NaB plus Torin1 (0.5μM; lower panel) for 24 hours and stained with anti-phospho KAP1 (S824) plus anti-ZEBRA antibodies and visualized at 1000X magnification. <b>C.</b> HH514-16 cells were transfected with scrambled siRNA or siRNA to <i>ATM</i>, treated with NaB after 24 hours and harvested after another 24 hours. Cell lysates were analyzed by immunoblotting with the antibodies indicated. Numbers below p-S824 KAP1 blot indicate relative amounts of pKAP1 after normalization to total KAP1. <b>D.</b> HH514-16-derived CLIX-FZ cells were exposed to doxycycline (upper panel) or doxycycline plus KU-55933 (lower panel) for 24 hours and stained with anti-p-S824 KAP1 plus anti-ZEBRA antibodies and visualized at 1000X magnification. <b>E.</b> Lymphoblastoid cells were transfected with pHD1013-Z plasmid and simultaneously treated with vehicle (-KU) or KU-55933. Treated cells were harvested 24 hours later and subjected to staining with anti-phospho KAP1 (S824) plus anti-ZEBRA antibodies and visualized at 1000X magnification. Experiments were performed twice.</p
Cellular KAP1 regulates EBV lytic cycle in Burkitt Lymphoma cells.
<p><b>A-C.</b> HH514-16 Burkitt lymphoma (BL) cells were transfected with scrambled siRNA (non-targeting control; open bars in B, C) or siRNA to <i>KAP1</i> (black bars in B, C); <b>D-F.</b> BL cells were transfected with empty vector (open bars in E, F) or pFLAG-CMV2-KAP1 (black bars in E, F). Transfected cells were treated with NaB 24 hours after transfection, and harvested at indicated times (A, D), 24 hours (B, E), or 48 hours (C, F) post-treatment to determine relative amounts of ZEBRA, KAP1 and β-actin by immunoblotting (A, D), relative levels of transcripts from EBV lytic genes <i>BZLF1</i>, <i>BMRF1</i> and <i>BFRF3</i> by qRT-PCR after normalization to 18S rRNA using the ΔΔC<sub>T</sub> method (B, E), and relative levels of cell-associated EBV DNA by qPCR (C, F). Error bars: SEM of 3 experiments with 4 technical replicates. Western blots are representative of 2 experiments. Numbers below blots indicate relative amounts of protein after normalization to β-actin.</p
KAP1 is phosphorylated at serine 824 during EBV lytic cycle in Burkitt lymphoma cells.
<p><b>A, D.</b> HH514-16 BL cells and HH514-16-derived CLIX-FZ cells were treated with increasing doses of NaB and doxycycline respectively for 24 hours, and lysates from cells were analyzed via immunoblotting with antibodies as indicated. <b>B, C, E, F.</b> HH514-16 cells were treated with NaB (B, C) or CLIX-FZ cells were treated with doxycycline (E, F) for 24 hours and stained with either anti-phospho KAP1 (S824) plus anti-ZEBRA antibodies (upper panels) or anti-phospho KAP1 (S824) plus anti-EA-D antibodies (lower panels) and visualized at 1000X magnification (B, E) or subjected to flow cytometry (C, F).</p
Phosphorylation of KAP1 at serine 824 impairs its ability to restrain EBV lytic cycle.
<p>HH514-16 BL cells were transfected with empty vector (EV in A; open bar in B and C), pFLAG-CMV2-KAP1 (KAP1 wt in A; black bar in B and C), pFLAG-CMV2-KAP1-S824A (KAP1 S824A in A; bars with horizontal lines in B and C) or pFLAG-CMV2-S824D (KAP1 S824D in A; bars with vertical lines in B and C), treated with NaB after 24 hours, and harvested immediately (0h) or after 48 hours for immunoblotting with antibodies as indicated (A), harvested after 24 hours for qRT-PCR to determine the relative levels of transcripts from EBV lytic genes <i>BZLF1</i>, <i>BMRF1</i> and <i>BFRF3</i> (B) or harvested after 48 hours to compare the relative levels of cell-associated EBV DNA by qPCR (C). Experiments were performed at least twice; error bars for B and C represent 3 technical replicates from 2 experiments.</p
Chloroquine induces phosphorylation of KAP1 at S824 and activates EBV lytic cycle in lymphoblastoid cells.
<p><b>A.</b> Lymphoblastoid cells were treated with chloroquine (CQ) and harvested at different times post-treatment for immunoblotting with antibodies as indicated. <b>B.</b> Lymphoblastoid cells were untreated (open bar), treated with 10μM CQ (black bar) or 100μM CQ (horizontal striped bar) every 3 days, released virus particles were pelleted from supernatant 9 days later, treated with DNase and quantified using q-PCR. ND: not detectable; error bars represent 3 technical replicates from 2 experiments.</p
NaB and chloroquine induce EBV replication without observable DNA damage.
<p><b>A.</b> HH514-16 cells were left untreated, treated with NaB or NaB plus KU-55933 for 24 hours or with etoposide for 16 hours and stained with anti-γH2AX plus anti-ZEBRA antibodies and visualized at 1000X magnification. <b>B.</b> HH514-16 BL cells were treated with chloroquine or chloroquine plus KU-55933 and harvested at 24 hours after treatment, stained for γH2AX and ZEBRA and visualized at 1000X magnification. <b>C.</b> Enlarged representative images of cells treated with NaB, chloroquine or etoposide. Experiments were performed twice.</p
Two new lignans with antioxidative activities from <i>Jatropha curcas</i>
<div><p>Activity-guided isolation of dried seeds of <i>Jatropha curcas</i> L. led to the isolation of two new lignans along with eight known compounds. These compounds were determined by spectroscopic analysis to be jatrophasin C (<b>1</b>), jatrophasin D (<b>2</b>), β-sitosterol (<b>3</b>), jatrophasin A (<b>4</b>), daucosterol (<b>5</b>), isoamericanol A (<b>6</b>), ( ± )-3,3′-bisdemethylpinoresinol (<b>7</b>), 7′-epi-sesamin-dicatechol (<b>8</b>), isoprincepin (<b>9</b>) and americanol A (<b>10</b>), of which <b>1</b> and <b>2</b> were new compounds. The antioxidative activities along with peroxisome proliferator-activated receptor gamma exciting activity of these compounds were also determined.</p></div
Additional file 1: of Overexpression of synuclein-ÃŽÅ‚ predicts lack of benefit from radiotherapy for breast cancer patients
Table S1. Prognostic factors of DMFS in univariate analysis of breast cancer patients were or were not treated with radiotherapy. Table S2. Independent predictors of DMFS in multivariate analysis of breast cancer patients were or were not treated with radiotherapy. (DOC 60 kb