Figure 6: The formation of centriole-like structures after LGALS3BP depletion depends on PLK4.

(a,b) Immunofluorescence microscopy and quantification of supernumerary centriole-like structures in U2OS cells after siRNA depletion of LGALS3BP and PLK4, as well as simultaneous depletion of both proteins. Knockdown of PLK4 in LGALS3BP-depletion background reveals a significant decrease of cells with >4 centrin-positive signals. (a) Immunofluorescence microscopy was conducted using antibodies against γ-tubulin and centrin. In the composite image γ-tubulin is shown in red, centrin in green and DAPI-stained DNA in blue. Scale bar, 5 μm for all images. (b) Data are expressed as means±s.d. of duplicates with n>200. (c–f) Immunofluorescence microscopy and quantification of supernumerary daughter centriolar marker CNTROB (c,d) and centrin (e,f) in U2OS cells after transient PLK4-TAP overexpression in the background of LGALS3BP depletion shows no difference in the formation of multiple procentrioles compared with non-targeting siRNA background and relevant controls. (c,e) PLK4-TAP was labelled with rabbit IgG shown in red, antibody-labelled CNTROB or centrin in green and DAPI-stained DNA in blue. Scale bars, 5 μm. (d,f) Data are expressed as means±s.d. of triplicates with n>200. (g) Immunofluorescence microscopy of stable myc-PLK4-U2OS cells after 24 h tetracycline induction in the background of LGALS3BP depletion shows no significant difference in number and localization of centrin and CNTROB compared with the background of non-targeting siRNA treatment. In the composite images, the labelling with specific antibodies is shown in green for centrin and in red for CNTROB. DAPI-stained DNA is shown in blue. The inserts at the bottom magnify the area of the centrioles. Scale bar, 5 μm. (h) The quantification of centriolar structures that are centrin-positive but centrobin-negative reveals no significant change after myc-PLK4 induction in a LGALS3BP-depletion background compared with the background of non-targeting siRNA treatment. Data are expressed as means±s.d. of duplicates with n>200. (i) Verification of the experiments in c–f by immunoblotting. Exogenous EGFP-TAP and PLK4-TAP is detected with rabbit IgG and LGALS3BP with a specific rabbit antibody. β-actin was used as loading control.