Figure 3: Knockdown of LGALS3BP leads to asterless spindle poles and multiple centrosomal structures.

(a) LGALS3BP siRNA treatment reveals its function for maintaining astral microtubules in mitotic U2OS cells. Furthermore, LGALS3BP-depleted cells have additional spindle pole-like structures (asterisks) that are detached from the main spindle microtubules. Depletion of positive control TUBGCP3 leads to bipolar asterless spindles. The negative control displays a normal spindle with two poles with aster microtubules after non-targeting siRNA treatment. The respective quantification of presence of astral microtubules is displayed in the chart. Data are shown as means±s.d. of duplicates with an average of n>90. (b) LGALS3BP depletion leads to supernumerary centrosome-like structures in mitosis, whereas TUBGCP3 siRNA treatment does not affect centrosome-like structure number significantly. The corresponding quantification of the centrosome-like structures is shown in the chart. Data are expressed as means±s.d. of duplicates with n>125 (a,b) Microtubules are labelled with an anti-α-tubulin antibody (green in the composite images in a); centrosome-like structures are labelled with an anti-γ-tubulin antibody (green in the composite images in b). Mitotic chromosomes are labelled with an anti-phosphorylated Histone H3 antibody (red in all composite images). DNA was labelled with DAPI (blue in all composite images). Scale bars, 5 μm. (c) Immunoblotting of U2OS cells treated with LGALS3BP and non-targeting siRNA shows siRNA efficiency and antibody specificity. β-actin was used as loading control.