Fig. 3.

Activation of α7 nAChR induces ROS production to promoteHIV-1transcription. (A) Volcano plot of upregulated and downregulated genes related to oxidative stress in the medium and GTS-21-treated ACH2 cells. (B) KEGG pathway analysis of upregulated and downregulated genes related to oxidative stress in the medium and GTS-21-treated ACH2 cells. Three samples in either medium or GTS-21 treated ACH2 groups were used for RNAseq analysis. (C) DCFH-DA fluorescent probes were used to label intracellular ROS, intracellular ROS level in untreated (Medium), GTS-21 (40 μM) treated and GTS-21(40 μM) &ROS scavenger N-Acetyl-L-methionine (NALM) (200 μM) treated groups by flow cytometry, cells without labeled with DCFH-DA probe were used as negative control (NC). (D, E) Detect the levels of p-p38 MAPK and gag transcription level in medium, GTS-21, GTS-21& NALM (200 μM) groups. (F) ACH2 cells were cultured with H₂O₂ (100 μM) for different times, and the level of p-p38 MAPK and gag were detected by western blotting. (G, H) Negative control and MAPK14 knockdown cells were cultured with H₂O₂ (100 μM) for 8, 12 h, (G) the levels of p-p38 MAPK and gag were detected by western blotting, (H) the expression level of gag mRNA was detected by RT-qPCR. Result is one representative from three independent repeats. Data are presented as mean ​± ​SD. ∗P ​< ​0.05, ∗∗∗∗P ​< ​0.0001 denote significant difference.