Figure 7: LGALS3BP knockdown rescues PCM dispersion in LGALS3BP endogenously overexpressing cells.

(a) LGALS3BP is highly expressed in the breast cancer cell line SK-BR-3 as compared with non-tumorigenic breast epithelial cell line MCF10A. Centrosomes are hypertrophic in SK-BR-3 cells, whereas centrosome morphology in MCF10A cells is normal. The siRNA depletion of LGALS3BP in SK-BR-3 cells reverts the centrosome dispersion phenotype, resembling normal centrosomes as in MCF10A cells. After pre-fixation extraction, the cells were labelled for PCM with an anti-γ-tubulin antibody shown in red in the composite image, LGALS3BP was labelled with a mouse antibody shown in green and DNA was stained with DAPI shown in blue in the composite image. Scale bars, 5 μm. (b) PCM hypertrophy of SK-BR-3 cells as opposed to normal centrosomes of MCF10A cells was quantified using an image analysis macro in ImageJ that determines the total area of PCM per cell. The diagram shows a comparison of the PCM area of MCF10A cells, SK-BR-3 cells and LGALS3BP-depleted SK-BR-3 cells, visualizing the decrease of PCM area and therefore a rescue of the PCM hypertrophy phenotype. The cells were tested in triplicates with each n>200. The percentage of cells that has been assigned to an interval of pixel numbers describing the area of the PCM for each cell type or knockdown including s.d. can be found in Supplementary Table S1. (c) Immunoblotting with a rabbit anti-LGALS3BP antibody shows that the SK-BR-3 cell line has a highly elevated level of LGALS3BP, whereas the MCF10A cell line has normal level of LGALS3BP (left panel). The endogenous level of LGALS3BP in MCF10A cells cannot be displayed to avoid complete overexposure of the blot through the SK-BR-3 sample. Therefore, exposure is only shown close to signal saturation for the SK-BR-3 cells. The right panel shows reduction of the endogenous LGALS3BP levels in SK-BR-3 cells after siRNA treatment corresponding to the experiments shown in b. β-actin (both panels) was used as a loading control.