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. 2020 Apr 23;117(19):10246–10253. doi: 10.1073/pnas.1913603117

Fig. 2.

Fig. 2.

The transcription factor CREB regulates CYP6CM1 expression. (A) Identification of the promoter of CYP6CM1 using progressive deletion constructs in dual luciferase reporter assays. S2 cells were cotransfected with pGL4.10 reporter plasmids carrying the indicated promoter regions conjugated to firefly luciferase and a reference reporter plasmid (pGL4.73, containing the hRluc reporter gene and an SV40 early promoter). Fluc/Rluc represents the ratio of Firefly to Renilla luciferase activity (n = 3, mean ± SE; ***P < 0.001, ANOVA with Tukey’s honestly significant difference [HSD] post hoc test). (B) Influence of 16 different B. tabaci transcription factors on expression driven by the promoter of CYP6CM1 in dual luciferase reporter assays. pGL4.10-CYP6CM1−939 to +1 and pGL4.73 were cotransfected into S2 cells with each of the 16 transcription factors in pAC5.1b. Empty pAC5.1b was used as a control (n = 3, mean ± SE; ***P < 0.001, ANOVA with Tukey’s HSD post hoc test). (C) Identification of important functional domains in CREB using dual luciferase reporter assays. pGL4.10-CYP6CM1−939 to +1 and pGL4.73 were cotransfected into S2 cells with pAC5.1b-CREB (CREB) with modified CREB in which the KID domain (CREB-DN1) or the bZIP domain (CERB-DN2) was deleted or with the pAC5.1b empty vector (control) (n = 3, mean ± SE; ***P < 0.001, two-tailed Student’s t test). (D) CREB binds to the upstream promoter sequences of CYP6CM1. EMSA results showing dose-dependent binding of CREB to a CRE-like–containing DNA probe. (E) Expression of CREB and CYP6CM1 protein after RNAi knockdown of CREB in the IMR strain as assessed by Western blot. Actin was used as a loading control. (F) Expression of CREB protein in the IMR and IMS strain as assessed by Western blot. Actin was used as a loading control. (G) Life span of adults of the IMR and IMS strains exposed to 2.0 mM imidacloprid (2) or no imidacloprid (0) after feeding on CREB dsRNA for 48 h. Adults fed on dsEGFP were used as a negative control.