Fig. 3.

Reporter assay analysis of targeted mutations in GC-boxes 1–3 and X-box. (A) Summary of point mutations made in luciferase reporter construct −232/+36. Wild-type nucleotide sequences encompassing predicted X-box and GC-box transcription factor binding sites (shaded) are shown above corresponding mutant sequences. All sequences are 5' to 3'; mutations are underlined. (B) Luciferase assays using PANC-1 cells transfected with wild-type or GC-box-mutant promoter constructs. GC-boxes are labelled 1 to 3; mutant GC-boxes are shaded black. Luciferase activity is expressed relative to wild-type. (C) Quantitative RT-PCR analysis of endogenous ALMS1 expression in PANC-1 cells before and after serum-starvation. (D) Primary cilium assembly in serum-deprived PANC-1 cells. Cells were incubated in growth medium containing either 10% or 0.5% FCS for 72 h and primary cilia visualized by immunostaining for acetylated tubulin (green), which labels ciliary axonemes and centrioles. Nuclear DNA was stained with DAPI (blue). Size bar, 10 µm. (E) Luciferase assays using PANC-1 cells transfected with wild-type or X-box-mutant promoter constructs. The mutant X-box is shaded black. Cells were incubated in either normal (10% FCS) or serum-starved (0.5% FCS) conditions for 48 h prior to lysis. Luciferase activity is expressed relative to wild-type. All data are from three independent experiments; error bars denote s.e.m.