Figure 3
Identification of intron 1 as the genomic region necessary for hyperosmotic GS induction. (a) The genomic sequences used for reporter assays are shown. The Ex1_3′ primer contains a NcoI restriction site at the 3′ end of exon 1. The grey bars indicate constructs that exclude intron 1 from the original constructs tested in Fig. 2 (black bars). The light green bar presents the functional GS core promoter that is used for constructing the backbone reporter vector GS-CP + 4.23. Three purple bars in the light green bar indicate downstream promoter elements (DPEs) that match to the ‘RGWYVT’ motif. (b) Hyperosmotic induction of reporter activity is completely abolished when intron 1 is excluded from the luciferase constructs. The normalized F/R ratio expresses inducible Firefly luciferase activity versus constitutive Renilla luciferase activity. This ratio was measured for both isosmotic (315 mOsmol/kg) and hyperosmotic (650 mOsmol/kg) conditions and normalized by setting isosmotic controls to one. One-way ANOVA was performed to assess statistical significance of the data and calculate p values using the R Stats package software (https://www.R-project.org/). The number of asterisks indicates the statistical significance of the hyperosmotic induction (***p < 0.001; **p < 0.01, *p < 0.05, ns not significantly different). Figure layout and abbreviations are as outlined in Fig. 2.
