Functional analysis of BmMet2 and kJHRE in mammalian HEK293 cells. HEK293 cells were treated as described below, and reporter activity was examined by using the Dual-Luciferase reporter assay system. Data represent means ± SD (n = 3). Means with the same letter are not significantly different (Tukey–Kramer test, P < 0.05). Some data were analyzed using Student’s t test (***P < 0.001; NS, P > 0.05). (A) Cells were cotransfected with a UAS reporter plasmid carrying firefly luciferase, a reference reporter plasmid (pRL-TK) carrying Renilla luciferase, and an expression plasmid carrying GAL4DBD fused with BmMet1, BmMet2, or VP16AD and were treated with 10 μM methoprene (JHA) for 24 h. (B) Cells were cotransfected with the UAS reporter plasmid and the expression plasmid carrying GAL4DBD fused with BmMet2 and were treated with the indicated concentrations of JH, JHA, MF, or FA for 24 h. (C) Cells were cotransfected with a kJHRE-reporter plasmid and an expression plasmid carrying native BmMet2 or BmSRC, and were treated with 0.1 μM JH I for 24 h. (D) Cells were cotransfected with a kJHRE-reporter plasmid, BmMet2 expression plasmid, and an expression plasmid carrying BmMet1, BmUSP, BmARNT, BmHIF-1α, or BmTimeless. (E) Cells were cotransfected with the UAS-reporter plasmid and an expression plasmid carrying GAL4DBD or VP16AD fused with BmMet2 or BmSRC and were treated with 0.1 μM JH I for 24 h. (F) A model for JH-mediated transcriptional induction of BmKr-h1. The physical interaction between kJHRE and BmMet2/BmSRC complex remains to be determined.