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Figure 6 | Retrovirology

Figure 6

From: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

Figure 6

Requirement of NUCKS1 for reactivation of provirus from HIV-1 latently infected cells. (A) Heatmap of mRNA expression of NUCKS1 and other Tat-interacting factors. (B) Expression level of endogenous NUCKS1 in HIV-1 latent cells (ACH-2 and J1.1) and their parent cell lines (A3.01 and Jurkat) were assessed with Western blotting using anti-NUCKS1 and anti-β-actin antibodies. The protein level of NUCKS1 was calculated by densitometry (Image Gauge Version 4.0, FujiFilm) (C) Normal and latently infected cells were treated with PMA (50 ng/ml) for indicated time. PMA-treated cell lysates were assessed by Western blotting using indicated antibodies. The levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). (D) mRNA-sequencing analysis. The RPKM (Reads Per Kilobase of exon model per Million aligned tags) was calculated for each transcript. The Y-axis represents the log2 fold changes between the control and PMA-treated ACH-2 cells. The right panel shows the expression level of NUCKS1 mRNA by RT-PCR. (E) ACH-2 and J1.1 cells were electrophorated with V5 or V5-NUCKS1. Three days later, the amount of virion in the supernatant was measured by HIV-1 p24 ELISA kit. The right panel shows the level of NUCKS1 expression by Western blotting. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to cells transfected with empty vector. (F-G) ACH-2 (F) and J1.1 cells (G) were electroporated with control or three individual NUCKS1 siRNAs. At 48 h after knockdown, the cells were treated with PMA (50 ng/ml) for 24 h and the levels of HIV-1 p24 were measured using an HIV-1 p24 ELISA kit. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared to the cells transfected with control siRNA.

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