Skip to main content
. 2014 Jan 14;33(2):129–145. doi: 10.1002/embj.201385946

Figure 1.

Figure 1

IQGAP1 is necessary for correct spindle orientation and single lumen formation.
  1. MDCK cells were transfected with three different siRNA heteroduplexes targeting canine IQGAP1. Transfected cells were grown in 3D cultures for 72 h and the efficiency of the siRNAs was analyzed in Western blots (WB).
  2. Quantification of IQGAP1 depletion. The siRNA#2 was most effective in silencing IQGAP1, resulting in 90% depletion, and it was therefore used in all the subsequent experiments (n = 3; values represent mean ± s.d.).
  3. MDCK cells were transfected with control or IQGAP1 siRNA and grown in 3D cultures. After 72 h the cysts were stained for Podxl (red), actin (green) and ToPro3 (blue). Scale bars, 5 μm.
  4. Single lumen formation was quantified with respect to that observed in control cysts. Only 59.5 ± 5.75% of siRNA#2 cysts polarized and formed a single lumen (n = 3; values represent mean ± s.d.).
  5. Control and IQGAP1 siRNA cysts were stained for actin (red) and acetylated tubulin (green). Scale bars, 5 μm.
  6. The angle formed between the mitotic spindle and the apicobasal axis was measured and the mean angles were: siControl = 75.53 ± 2.69°; siIQGAP1 = 45.47 ± 4.57° (n = 3, >40 cysts per experiment, values represent mean ± s.e.m.).
  7. Scheme of the experimental design (left panel). Control cells and IQGAP1 KD cells were grown in 3D cultures immediately after nucleofection. After 4 days, control cells organized in epithelial organoids surrounding a central lumen (group G1), while IQGAP1-KD cysts displayed defects in single lumen formation when compare to the controls (group G3). Control cells treated with thymidine (2 mM), formed smaller cysts with single lumens in the same rate as those not treated with the mitotic inhibitor (group G2). After 48 h, IQGAP1 interference starts to be effective, although the addition of thymidine at this point completely recovered normal lumen formation (group G4: Right panel) Representative images of cysts grown for 4 days under the conditions described previously. Scale bars, 5 μm.
  8. Single lumen quantification under each condition described in (G). Cysts forming single lumen: group G1 = 73.66 ± 1.23%; group G2 = 71.36 ± 7.84%; group G3 = 52.16 ± 5.13%; group G4 = 69.98 ± 6.19% (n = 3, >100 cysts per experiment; values represent mean ± s.d.). Error bars represent the s.e.m. or s.d.: *P < 0.05; **P < 0.01.

Source data are available online for this figure.