|
|
Blood, Vol. 113, Issue 21, 5192-5201, May 21, 2009
HIV-1 transactivator protein induction of suppressor of cytokine signaling-2 contributes to dysregulation of IFN signaling
Blood Cheng et al.
113: 5192
Supplemental materials for: Cheng et al
Plasmid information
Expression vector (pcDNA3.0) and the luciferase reporter vector (pGL3) were purchased from Invitrogen (Grand Island, NY) and Promega (Madison, WI), respectively. The cDNA containing eight copies of the GAS responsive element (8×GAS) was kindly provided by Dr. Minho Shong (Chungnam National University). Antibodies
Antibodies against STAT1, pSTAT1 (Tyr701), and pSTAT1 (Ser 727) were purchased from Cell Signaling Technology (Beverly, MA). The anti-actin or anti-Lamin B1 antibodies and anti-SOCS-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Zymed (Grand Island, NY), respectively. Antibodies against Tat was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Monoclonal Antibody to HIV-1 Tat (NT3 2D1.1) from Dr. Jonathan Karn, courtesy of the NIBSC Centralised Facility for AIDS Reagents. Reagents for cell culture and TRIzol were obtained from GIBCO/BRL (Grand Island, NY). Transfection reagent, jetPEI, was purchased from PolyPlus (San Marcos, CA). Nuclear extract preparation
PBMo (106 cells) were resuspended in 200 µl of buffer A (10 mM Hepes; pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) on ice for 15 minutes. This was followed by the addition of NP-40 to a final concentration of 0.2% for 2.5 minutes for plasma membrane lysis. Nuclei were obtained by centrifugation at 13,000 rpm, and nuclear proteins were extracted in 50 µl of buffer C containing 20 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 300 mM NaCl, 0.5 mM DTT, 0.2 mM PMSF, and 20% glycerol. Recombinant SOCS-2 production
Expression of SOCS-2 was induced by adding 0.1 mM IPTG to Escherichia coli strain BL21(DE3)pLyS and incubated for 5 hours at 37°C. The bacterial cells were lysed by sonication, and the cell debris was removed by centrifugation. SOCS-2 protein in the soluble fraction was purified using His-Bind columns (Novagen), and the purified protein was further concentrated using Ultra centrifugal filter devices of MWCO 10,000 (Millipore, Bedford, MA). IL-10 siRNA study
IL-10 knockdown in CD14+ PBMo was accomplished by using IL-10 specific siRNA (Stealth™, Invitrogen) and the Stealth™ RNAi Negative Control Duplexes (Low GC Duplex, Invitrogen). These sequences contain no cross-homology to other sequences in the GenBank database. Transfection of the primary cells was conducted using jetPEI reagent (polyethyleneimine, PolyPlus). CD14+ PBMo (1 × 106 cells/ml in 1 ml) were transfected with 100 µl of jetPEI-siRNA complex (2 µl: 100 nM) for 24 hours and then treated with 10 nM of Tat for 5 hours. For the protein harvest, of IFN (100 U/ml) was added for 10 minutes after 5 hours of Tat pretreatment. QRT-PCR of HIV-1 Tat
Total cellular RNA extraction and the cDNA synthesis were as described in our previous reports.18,19 The cDNA produced were then subjected to QRT-PCR assays using HIV-1 Tat specific primers.
Files in this Data Supplement:
- Figure S1. Effects of Tat on the serine phosphorylation of STAT1, and the levels of endotoxicity as well as cytotoxicity of Tat protein (JPG, 100 KB)
-
(A) PBMo were untreated (UT), mock-treated (M) or treated with indicated concentrations of Tat (T) for 4 hours and followed by the treatment of IFN (100 U/ml) for 10 minutes. The serine phosphorylation of STAT1 (Ser 727) was probed with specific antibodies as indicated. Total STAT1 was probed as a loading control. (B) The endotoxicity of indicated concentrations of Tat was measured by Pyrotell assay. (C) Viability of Tat treated primary blood macrophages. Primary blood macrophages were incubated with Tat (10nM) at indicated time. The PI stained cells were measured by immunofluorescence imaging (Thermo Scientific Cellomics ArrayScan VTI Live, Pittsburgh). All data presented were plotted as mean values ± standard error of mean (S.E.M.) of three independent experiments using three independent donors’ cells. (D) PBMo were treated with indicated concentrations of LPS and SOCS2 mRNA levels were measured by QRT-PCR. (E) PBMo transfected with either control siRNA or IL-10 siRNA were pretreated with Tat for 5 hours and followed by IFNγ treatment for 10 minutes. The phosphorylation levels of STAT1 were examined by Western blotting using anti-pSTAT1 antibodies. Total STAT1 was probed as a loading control. (F) To confirm the efficiency of IL-10 siRNA in CD14+ PBMo, cells were transfected with either control siRNA or SOCS-2 siRNA, and later treated with Tat for 5 hours as indicated. The siRNA-mediated IL-10 knockdown in PBMo was verified using RT-PCR (lane 4 versus 5). (G) Primary blood macrophages were treated with HIV-pseudovirus or with plasmids alone (mock) for 48 hours and cellular RNA were collected. The HIV-1 Tat mRNA copies were measured by RT-PCR using HIV-1 Tat specific primers and normalized with known copies of Tat.
- Figure S2. The expression levels of SOCS-1, -2, and -3 mRNA in PBMo (JPG, 110 KB)
-
(A) PBMo were treated with 10 nM of Tat for indicated time intervals. The expression levels of SOCS-1, -2, and -3 transcripts were examined using RT-PCR. GAPDH was used as a loading control. (B) PBMo were treated with 10 ng/ml of recombinant human IL-10 for indicated time intervals. The expression levels of SOCS-1, and -3 transcripts were examined using RT-PCR. (C) SOCS-1 and SOCS-3 expression in HIV-pseudovirus treated macrophages. Primary blood macrophages were treated with HIV-pseudovirus at the indicated m.o.i., Tat (10nM) or the control plasmid (pNL4-3) for 48 hrs. Total RNA samples were harvested for the measurement of SOCS-1 and SOCS-3 transcripts using RT-PCR. GAPDH was used as a loading control. A representative figure of 3 independent experiments on macrophages from three different donors is shown. (D) HIV-pseudovirus treatment inhibited IFN-γ induced STAT1 (Tyr 701) phosphorylation. Primary blood macrophages were treated with either HIV-pseudovirus, control plasmid or Tat for 48 hours. After that, IFN-γ (100U/ml) was added to the virus-treated macrophages for 10 minutes. Total cellular protein samples were collected and examined by Western blotting using anti-pSTAT1 (Tyr 701) or anti-STAT1 antibodies. A representative figure of 3 independent experiments on macrophages from three different donors is shown. Vertical lines have been inserted to indicate a repositioned gel lane.
- Figure S3. Expression and purification of recombinant SOCS-2, and Western analysis of Tat-induced SOCS-2 (JPG, 69.7 KB)
-
(A) E.coli, BL21(DE3)pLyS was transformed with pET28a-SOCS-2 expression vector, while recombinant SOCS-2 protein expression was induced by IPTG. The expression of SOCS-2 was confirmed by Western blotting using monoclonal anti-His6 antibodies (lane 2 to 4). The recombinant SOCS-2 protein was purified using Ni+-charged column (lane 6 to 8). Purified protein was verified by Western blotting using specific anti-SOCS-2 antibodies (lane 9). (B) PBMo were untreated (UT), Tat-treated (T) or Mock-treated (M) for the indicated period of time. Cell lysates of different amount were loaded for Western blotting using anti-SOCS-2 antibodies. Actin was probed as a loading control.
- Figure S4. Expression of SOCS-1, -2, and -3 in HEK293 cells overexpressing SOCS-2, and in different monocytic cell lines (JPG, 62.7 KB)
-
(A) HEK293 cells were either un-transfected (UT) or transfected with plasmid pRL-TK (transfection control), pGL3-8×GAS (luciferase reporter vector), pcDNA (parental vector), pcDNA-SOCS-2 (SOCS-2 expression vector), and All (all components of reporter assay) for 24 hours. The expression levels of SOCS-1, -2, and -3 were examined by RT-PCR. (B) The endogenous expression levels of SOCS-1, -2, and -3 among monocytic cell-lines were examined by RT-PCR as indicated. GAPDH was used as a loading control.
|
|
