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Fig. 2 | Virology Journal

Fig. 2

From: Activation of HIV-1 expression in latently infected CD4+ T cells by the small molecule PKC412

Fig. 2

Pulse PKC412 treatment stimulates HIV-1 expression in ACH2 cells. ACH2 cells were pulse-treated with PKC412 (0.5 μM) for 8, 12, 16, 24, or 48 h. PMA (2 ng/ml) or DMSO-treated cells were used as the positive and negative controls, respectively. a After 24 h, the cells were fixed and labeled with an anti-HIV-1 p24 antibody/anti-mouse IgG-FITC antibody and visualized under the fluorescence microscope (10× magnification). b After 48 h, the cells were lysed and analyzed by SDS-PAGE followed by Western blotting with anti-HIV-1 gp120, anti-HIV-1 p24, and anti-tubulin antibodies. c The HIV p24 levels in supernatants were quantified using an HIV-1 p24 ELISA kit after 48 h. d ACH2 cells were incubated with RPMI medium (1 % FBS) containing different PKC412 concentrations. After 48 h, total RNA was extracted from the PKC412-treated or untreated ACH2 cells and HIV transcription was measured by real-time PCR using primers corresponding to the HIV 5′LTR and gag (R-gag). Transcription activity was calculated as the relative HIV-1 mRNA level by setting the HIV-1 mRNA level in the control ACH2 cells (without PKC412 treatment) to 1 arbitrary unit. Error bars represent variations between triplicate samples and the data are representative of results obtained in three independent experiments. The degree of significance for PKC412 treatment was relative to DMSO treatment. * p < 0.05, ** p < 0.01

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