Figure 4: Depletion of LGALS3BP leads to supernumerary centriolar structures.

(a) siRNA-mediated depletion of LGALS3BP leads to multiple centriolar structures as identified with an anti-centrin antibody both in interphase and in mitotic U2OS cells. There are multiple centrin-positive structures visible that are partly not associated with PCM as marked with an anti-γ-tubulin antibody. The inserts for each image magnify the area of the centrosome. In the composite images, γ-tubulin is displayed in red, centrin in green and DAPI-stained DNA in blue. Scale bars, 5 μm. (b) Quantification of centriolar structures per centrosome. Data are shown as means±s.d. of duplicates with n>200 for each experiment. (c) Centriolar structures resulting from LGALS3BP depletion contain centriolar proteins CPAP and acetylated tubulin in addition to centrin as shown by antibody labelling. CPAP and acetylated tubulin are coloured in red, centrin in green and DAPI-labelled DNA in blue in the composite images. Scale bars, 10 μm. (d) TEM of U2OS cells treated with LGALS3BP siRNAs reveals abnormal centriolar structures indicated by arrowheads. Microtubules emanating from centriolar structures are marked by asterisks. Scale bars, 200 nm. (e) Quantification of centriolar structures determined by TEM with n=37 for non-targeting and n=50 for LGALS3BP siRNA treatment. (f,g) Depletion of endogenous LGALS3BP using a siRNA pool targeting the 3′ untranslated region (3′UTR) of LGALS3BP with simultaneous overexpression of exogenous LGALS3BP-YFP up to 96 h decreases the supernumerary centriolar structures up to control ranges in U2OS cells (immunoblotting in Supplementary Fig. S1). (f) Quantification of the centriolar structures in the LGALS3BP-rescue experiment. Data are expressed as means±s.d. of triplicates with each n>200. (g) Cells depleted for endogenous LGALS3BP show normal centriole number when LGALS3BP-YFP is expressed demonstrating the rescue of centriolar LGALS3BP-depletion phenotypes. This is not the case in cells expressing EGFP-YFP as control. The composite images show LGALS3BP-YFP and EGFP-YFP in red, centrin in green and DAPI-labelled DNA in blue. The asterisk indicates a LGALS3BP-YFP-transfected cell with rescue adjacent to an untransfected cell with supernumerary centriolar structures. Scale bars, 10 μm.