Figure 5: Intracellular CD24 and proliferation of prostate cancer cells.

(a) Measurement of cell surface and total cellular CD24 by flow cytometry. The prostate cancer cell lines PC3, DU145 and LNCaP were stained with anti-CD24 with or without permeabilization to measure either cell surface (top) or total cellular CD24 (bottom). (b) Analysis of CD24 in the cytoplasmic and plasma membrane fraction (Cp+Cm), nucleoplasm (Np) and the insoluble fraction that includes chromatin by western blot. The efficiency of the fractionation was monitored by the presence of β-actin (Cp+Cm+Np), Lamin (Np) and H1.5 (chromatin). (c) Confocal microscopy reveals dynamic upregulation and localization of CD24 during mitosis. Note alterations of CD24 distribution and signal intensity at different phases during mitosis. See Supplementary Fig. 6 for single-colour images. (d) Upregulation of CD24 during mitosis as revealed by western blot of cellular lysates prepared from nocodazale-synchronized DU145 cells. (e) Diagram of CD24-GFP and GFP-CD24 constructs. The N-terminus-tagged CD24 was produced by inserting the GFP-coding sequence after the signal peptide, whereas the C-terminus-tagged CD24 was generated by inserting the GFP-coding sequence before the stop codon of CD24. (f) Tagging the C-terminus with GFP prevents cell surface localization of CD24. The images show the distinct distribution of green fluorescence in cells transfected with either CD24-GFP (C) or GFP-CD24 (N). (g) Verification of cell surface versus intracellular CD24 expression by cell surface staining. CD24-GFP (C) was not present on the surface of DU145 cells as it was not detected by an anti-CD24 mAb added to non-permeabilized cells. CD24-GFP (N) was used as positive control for cell surface CD24. (h) Verification of fusion protein expression by western blot using anti-CD24 and anti-GFP antibodies. (i) Intracellular CD24 encoded by CD24-GFP (C) is at least as potent in promoting proliferation of DU145 cells ad GFP-CD24 (N). CD24-silenced DU145 cells were transfected with CD24-GFP (C) or GFP-CD24 (N) plasmids. After drug selection, an equal number of drug-resistant transfectants were plated onto 10-cm plates. The colonies were stained with crystal violet. All images have been reproduced three times.