Figure 4.

IQ motifs restrict IQGAP1 to the basolateral membrane.

1. Scheme showing the constructs used, each designed as a GFP fusion protein.
2. MDCK cells stably expressing IQGAP1-ΔIQm eGFP were transfected with the control or IQGAP1 siRNA and grown in 3D cultures to form cysts, which were fixed and co-stained for actin (red) and β-catenin (blue). The basolateral (arrows) and apical (arrowheads) localization of the mutant construct is indicated.
3. The ability of the IQGAP1-ΔIQm stable clone to form single lumen cysts was quantified and represented as the percentage of the total number of cysts: Control, 98 ± 1.4%; IQGAP1-ΔIQm, 61.5 ± 7.4%; siIQGAP1, 59.5 ± 7.05%; IQGAP1-ΔIQm + siIQGAP1, 46.2 ± 4.18% (n = 3, >50 cysts per experiment; values represent the mean ± s.d.).
4. MDCK IQGAP1-ΔIQm eGFP cysts were stained for acetylated tubulin (red) and the yellow line represents the mitotic spindle angle.
5. The angle formed between the mitotic spindle and the apicobasal axis was measured and the mean angles were: control = 76.10 ± 2.29°, IQGAP1-ΔIQm = 43.09 ± 4.40° (n = 3, 40 cells per experiment; values represent the mean ± s.e.m.).
6. MDCK IQGAP1-ΔIQm eGFP cysts were treated with PMA for 15 min, fixed and co-stained for actin (red) and β-catenin (blue). The basolateral (arrows) and apical (arrowheads) localization of the mutant construct is indicated.

Data information: Error bars represent the s.d. or s.e.m.: *P < 0.05; **P < 0.01. Scale bars, 5 μm.