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Results: 5

1.
Figure 3

Figure 3. NF-κB signaling is necessary for METH-mediated induction of HIV-LTR-Luc-IRES-GFP. From: Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

CHME-5 cells were co-transfected with pLTR-Luc-IRES-GFP and pI-κBDN, and treated with 500 µM of METH. Luciferase activity was measured at 24 h post-treatment. IκBDN significantly decreased luciferase activity. (p<0.001 One-way ANOVA, *** p<0.001 SNK post-hoc versus other groups).

Emily S. Wires, et al. J Neurovirol. 2012 October;18(5):400-410.
2.
Figure 5

Figure 5. Nuclear translocation of NF-κB in HIV-latently infected CHME-5 cells treated with METH. From: Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

A, Reactivation of CHME-5/HIV by METH. Cells were incubated either in the absence (untreated) or in the presence of METH (600 µM) for 30 minutes, 16 h and 24 h as indicated next to each set of Brightfield and GFP-fluorescence pictures. B, NF-κB p65 Western blot of nuclear extracts isolated from untreated and METH-treated cells. MW markers and time of treatment are indicated above the blots; the weight of markers is indicated in KDa (red bands), and the presence of proteins is indicated by the green bands. SPT-5 and TFIIH (indicated to the right of the corresponding blot) were used as control.

Emily S. Wires, et al. J Neurovirol. 2012 October;18(5):400-410.
3.
Figure 2

Figure 2. METH increases activity of cellular transcription factors in CHME-5 cells. From: Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

A, CHME-5 cells were transfected with a reporter plasmid containing minimal promoter with NF-κB response elements, and treated with METH for 24 h. METH treatment significantly increased luciferase activity (One-way ANOVA Dunnet Test post-hoc versus 0 METH, *p<0.05, ***p<0.001). B, CHME-5 cells were treated with 500 µM METH for various times. Nuclear extracts were prepared and assayed for NF-κB p65 by ELISA. NF-κB p65 was significantly elevated in nuclear extract as early as 30 minutes and remained elevated at 24 h. LPS treatment for 1 h was used as a positive control, (p<0.001, One-way ANOVA Dunnet post-hoc versus 0 METH, **p<.01,***p<0.001). C, CHME-5 cells were treated with various amounts of METH for 1 h. Nuclear extracts were prepared and assayed for p65 by ELISA. NF-κB was significantly elevated in nuclear extracts treated with 500 µM, 700 µM, and 1000 µM METH. (p=0.01, One-way ANOVA Dunnet post-hoc versus 0 METH, **p<0.01).

Emily S. Wires, et al. J Neurovirol. 2012 October;18(5):400-410.
4.
Figure 1

Figure 1. METH induces luciferase and GFP expression from HIV-LTR-Luc-IRES-GFP in CHME-5 cells. From: Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

A, CHME-5 cells were co-transfected with pLTR-Luc-IRES-GFP reporter together with a control (−Tat) or Tat-expressing plasmid (+Tat). Cells were treated with METH for 4 h post-transfection and assayed for luciferase activity at 24 h post-transfection. METH significantly increased activity independently of Tat (*p<0.05, **p<0.01, ***p<0.001, One-way ANOVA SNK post-hoc versus 0 METH); (# p<0.001, Student t-test versus No Tat, 0 METH). B, CHME-5 cells were transfected with pLTR-Luc-IRES-GFP together with pC-Tat and treated with 0, 500, and 700 µM of METH for 24 h and stained with mouse anti GFP (indicated to left of the Brightfield panels). Brightfield (left panel) and GFP fluorescence (right panel) were imaged for each treatment group. METH increased GFP fluorescence at 500 and 700 µM METH compared to vehicle-treated cells. C, METH did not significantly alter viability (MTS assay) of CHME-5 cells treated with indicated doses of METH.

Emily S. Wires, et al. J Neurovirol. 2012 October;18(5):400-410.
5.
Figure 4

Figure 4. METH reactivates CHME-5 cells latently infected with HIV. From: Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

A, Genomic organization of the HIV lentiviral vector. A fragment of HIV-1pNL4-3, containing Tat, Rev, Env, Vpu, and Nef with the reported gene d2EGFP, is cloned into the pHR’ backbone. The resulted plasmid was used to produce the VSVG HIV as described previously (Kim et al, 2006). B Reactivation of HIV by METH. CHME-5 cells were latently infected with HIV (CHME-5/HIV) and incubated either in the absence (untreated) or in the presence of increasing doses of METH, as indicated above each set of brightfield and GFP-fluorescence pictures. Cells were reactivated with TNFα (50 ng/mL), as positive, control. FACS plots show increase in the % of GFP+ cells in response to METH or TNFα. C, Reactivation of HIV by METH depends on the activation of NF-κB. CHME-5/HIV cells were pre-incubated with 100 µM of either an inhibitory of the IKK complex (Ikka/Ikkb) or a control peptide (ctl peptide) prior to incubation with METH (600 µM).

Emily S. Wires, et al. J Neurovirol. 2012 October;18(5):400-410.

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