The CHME-5 cell line was created by transfecting human fetal microglia with the large T antigen of the simian virus 40 (Janabi et al, 1995 (link)). Cells were maintained in Dulbecco’s minimal essential medium, high glucose (DMEM HG) supplemented with 5% FBS and 1% penicillin/streptomycin based on previous studies (Cox et al, 2009 (link)).
For transfection, cells were plated in clear or opaque 96 well plates (Corning Costar, Sigma Aldrich) at 8×104 cells/well and transfected 14–16 h post-plating using Lipofectamine 2000 (Invitrogen, CA). For luciferase experiments, pLTRC-Luc-EGFP and Tat expressing plasmids were kept constant and, 4 h after transfection, METH was added at indicated concentrations. Cells were assayed 24 h post-transfection unless otherwise noted.
For obtaining HIV-latently infected CHME-5, cells were plated on a 6-well plate at a density of (1×106 cells/well) for 48 h prior to infection with vesicular stomatitis virus G-(VSVG) pseudotyped HIVs bearing a fragment of HIV-1pNL4-3, containing Tat, Rev, Env, and Vpu, cloned into the pHR' backbone (Dull et al, 1998; Pearson et al, 2008), plus Nef adjacent to the reporter gene d2E green fluorescence protein (GFP) inserted next to Env. The viral particles were produced by the triple transfection of 293T cells using lipofectamine, as described previously (Kim et al, 2006 (link)), and the vector titer was determined by the infection of 1×106 CHME-5 cells with a serial dilution of the harvested medium supernatant. Briefly, the plate containing cells and virus was spinoculated in a swing-bucket centrifuge at 3000 × g for 1.5 h at room temperature, and incubated at 37°C in 5% CO2 for 48 h prior to trypsin treatment and fluorescence-activated cell sorting (FACS). GFP+ cells were further cultured and allowed to enter into a latent state (GFP expression below 5%) for four weeks. Latency of CHME-5/HIV cells was characterized by re-activating HIV expression (GFP; see below) and evaluating nuclear translocation of NF-κB p65 and two of its phosphorylated forms (S536 and S468) by Western blot (see below) in the presence of TNFα (50 ng/mL) for 0.5, 2, 4, 8, and 16 h. Latent CHME-5/HIV cells were then untreated or treated with increasing concentration of METH (0, 50 and 300 µM). GFP fluorescence was imaged using identical acquisition setting among groups within a given experiment. Images were acquired using a Nikon TE2000 inverted scope equipped with a DS-QiMc camera and controlled by NIS Elements software (Nikon). Again, treatment with 50 ng/mL TNFα (Pearson et al, 2008) was used as a positive control for reactivation. For testing NF-κB dependence of METH-mediated activation of HIV, CHME-5/HIV cells were pre-treated for 1 h with either 100 µM of IKKγ NEMO binding domain inhibitory peptide or equivalent amount of the control peptide (Imgenex, CA) dissolved in dimethyl sulfide (DMSO) prior to incubation with 600 µM of METH for 16 h.
For transfection, cells were plated in clear or opaque 96 well plates (Corning Costar, Sigma Aldrich) at 8×104 cells/well and transfected 14–16 h post-plating using Lipofectamine 2000 (Invitrogen, CA). For luciferase experiments, pLTRC-Luc-EGFP and Tat expressing plasmids were kept constant and, 4 h after transfection, METH was added at indicated concentrations. Cells were assayed 24 h post-transfection unless otherwise noted.
For obtaining HIV-latently infected CHME-5, cells were plated on a 6-well plate at a density of (1×106 cells/well) for 48 h prior to infection with vesicular stomatitis virus G-(VSVG) pseudotyped HIVs bearing a fragment of HIV-1pNL4-3, containing Tat, Rev, Env, and Vpu, cloned into the pHR' backbone (Dull et al, 1998; Pearson et al, 2008), plus Nef adjacent to the reporter gene d2E green fluorescence protein (GFP) inserted next to Env. The viral particles were produced by the triple transfection of 293T cells using lipofectamine, as described previously (Kim et al, 2006 (link)), and the vector titer was determined by the infection of 1×106 CHME-5 cells with a serial dilution of the harvested medium supernatant. Briefly, the plate containing cells and virus was spinoculated in a swing-bucket centrifuge at 3000 × g for 1.5 h at room temperature, and incubated at 37°C in 5% CO2 for 48 h prior to trypsin treatment and fluorescence-activated cell sorting (FACS). GFP+ cells were further cultured and allowed to enter into a latent state (GFP expression below 5%) for four weeks. Latency of CHME-5/HIV cells was characterized by re-activating HIV expression (GFP; see below) and evaluating nuclear translocation of NF-κB p65 and two of its phosphorylated forms (S536 and S468) by Western blot (see below) in the presence of TNFα (50 ng/mL) for 0.5, 2, 4, 8, and 16 h. Latent CHME-5/HIV cells were then untreated or treated with increasing concentration of METH (0, 50 and 300 µM). GFP fluorescence was imaged using identical acquisition setting among groups within a given experiment. Images were acquired using a Nikon TE2000 inverted scope equipped with a DS-QiMc camera and controlled by NIS Elements software (Nikon). Again, treatment with 50 ng/mL TNFα (Pearson et al, 2008) was used as a positive control for reactivation. For testing NF-κB dependence of METH-mediated activation of HIV, CHME-5/HIV cells were pre-treated for 1 h with either 100 µM of IKKγ NEMO binding domain inhibitory peptide or equivalent amount of the control peptide (Imgenex, CA) dissolved in dimethyl sulfide (DMSO) prior to incubation with 600 µM of METH for 16 h.