Fig 1: The MUC5B –3 kb region exhibits enhancer activity that is regulated by STAT3 and SPDEF.(A) Location of MUC5B Short (red, 766 bp) and Long (blue, 4532 bp) regions cloned into reporter constructs relative to the transcription start site (black arrow). (B–F) Mean (±SD) normalized luciferase activity of indicated Short (red; top) and Long (blue; bottom) MUC5B reporter constructs and respective empty vector (EV) control in A549 cells. (B) WT constructs under basal culture conditions (n = 4/group, *P = 0.05 versus EV via t test). (C) WT versus mutant constructs with binding site mutations for STAT3, ETS1, or both (n = 4/group, *P = 0.0001 versus WT via 1-way ANOVA with Bonferroni correction). (D) WT constructs cotransfected with SPDEF or STAT3 cDNA expression constructs or empty expression vector control (Ctrl; n = 4/group, *P = 0.003 versus WT + Ctrl via 1-way ANOVA with Bonferroni correction). (E and F) WT constructs cotransfected with siRNA targeting SPDEF (E), STAT3 (F), or control (siCtrl; n = 4/group, *P = 0.001 versus WT + siCtrl via 1-way ANOVA with Bonferroni correction). (G) ChIP-qPCR analysis of mean (±SD) relative occupancy of SPDEF and IgG across the MUC5B –3 kb region in A549 cells (n = 3 (IgG) or 4 (SPDEF) per group; *P = 0.05 versus geometric mean of IgG occupancy at 3 negative control regions via t test. Schematic indicates locations of putative STAT/ETS1 binding sites and regions targeted by ChIP-qPCR primers. (H) Mean (±SD) normalized luciferase activity of WT and mutant MUC5B Short reporter constructs with G or variant T at the G/T transversion site, respectively (n = 4/group). (I) qPCR analysis quantifying relative MUC5B expression in untransfected WT control (Ctrl-GG) and CRISPR-Cas9–edited WT (GG) and variant (TT) A549 clones (n = 3/group). Data in each panel are representative of at least 3 independent experiments, except for I, which was performed in 2 independent clones for each genotype.