FIG 6.
HML-2 knockdown reduces ISRE activation through direct signaling and distantly via a paracrine mechanism. (A to F) Reverse transcriptase quantitative PCR (RT-qPCR) measured expression (threshold cycle [ΔΔCT] method) of genes IRF1, SOCS3, and ICAM1 which contain a GAS element (A to C) and an ISRE element-containing genes IFIT1, ISG15, and OASL (D to F) in response to IFN-γ treatment for 18 h in TDMs transduced with control shRNA or HML-2-shRNA targeting a conserved region of the HML-2 env gene transcript. (G) Measuring direct activation of ISRE elements in response to IFN-γ treatment using luciferase-reporter THP1-Dual or THP1-Dual MAVS-KO (mitochondrial antiviral-signaling protein-knockout)-derived macrophages transduced with either control-shRNA- or HML-2-shRNA-expressing vectors; 18 h post-IFN-γ treatment. (H) Measuring paracrine activation of ISRE elements in luciferase reporter THP1-Dual-derived macrophages in response to condition medium from TDMs transduced with control shRNA- or HML-2-shRNA-expressing vectors and treated with IFN-γ for 18 h. Data are presented as means ± SEM from three independent measurements in panels A to F and three biological replicates in panels G and H. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 according to two-way ANOVA.