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Figure 2 | Journal of Neuroinflammation

Figure 2

From: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages

Figure 2

Relative gene expression levels of the Hutat2:Fc and EGFP genes in transduced cells and quantification of Hutat2:Fc in conditioned mediums. (A) Detection of Hutat2 and EGFP mRNA in HR-Hutat2 transduced cells by a RT-PCR qualitative analysis. HTB-Hutat2, HR-Hutat2 transduced HTB-11 RNA; U937-Hutat2, HR-Hutat2 transduced U937 RNA; hMDM-Hutat2, HR-Hutat2 transduced hMDM RNA; U937, non-transduced U937 RNA; IC, Internal control RNA from K562 cell line; NTC, No template control; RTase (-), RTase negative control. (B and C) Quantitative real-time PCR analysis of Hutat2 and EGFP gene expression levels in transduced HTB-11 and U937 cells compared with that in transduced hMDM (*P <0.01). (D) Comparison of Hutat2:Fc secretion level between transduced HTB-11 and U937 within 24 hours (*P <0.01); 1 × 106 cells were plated into a T-75 flask and the mediums were collected 24 hours later. Hutat2:Fc was quantified by a human IgG ELISA method. (E and F) Detection of Hutat2:Fc proteins in cell lysate and supernatant of transduced cells by Western blotting. (G) Detection of stable secretion of Hutat2:Fc in conditioned mediums from HR-Hutat2 transduced HTB-11 (HTB-Hutat2) and U937 cells (U937-Hutat2). Cells were passaged totally 20 times and an ELISA assay was performed every fifth passage. (H and I) The accumulation of Hutat2:Fc in mediums from transduced HTB-11 and U937 cells; 1 × 106 cells were plated into a T-75 flask and the mediums were collected every 24 hours for 4 days. (J) Kinetics of Hutat2:Fc levels in cell culture supernatants of transduced hMDM at different MOI after transduction. The levels of secreted Hutat2:Fc were peak on day 9 post-transduction. The concentrations of Hutat2:Fc were higher at MOI 50 than at MOI 10 in mediums of transduced hMDM at each time point (P <0.01). Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.

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