Figure 3

Characterization of NT and SUSD2-KD HGSOC cell lines. (a) Flow cytometry analysis. Flow cytometry analysis was performed on HGSOC cells (OVCAR3, OVSAHO and KURAMOCHI cell lines) using an anti-SUSD2 antibody. All HGSOC cell lines shown were transfected with either an NT shRNA construct (abbreviated ‘NT’; shown in red) or one of two SUSD2-targetting shRNA constructs (abbreviated ‘sh1/2/4/1-2/4-4’; shown in shades of blue). The black curves in each graph depicts the background fluorescence observed when nonspecific binding of the secondary antibody alone binds to cell surface proteins. Fluorescence is shown in the x axis and the number of cells is shown in the y axis. (b) Analysis of SUSD2 levels in HGSOC whole-cell lysates. The upper two panels show protein bands from western (WB) immunoblot analysis detecting SUSD2 and GAPDH in whole-cell lysates. In the lower panel, immunoprecipitation (IP) reactions on HGSOC whole-cell lysates were performed using an anti-SUSD2 antibody. Immunoprecipitated proteins were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel followed by WB analysis using an anti-SUSD2 antibody. The band representing full-length, post-translationally modified SUSD2 is ~100 kDa. The 90 kDa band represents partially glycosylated SUSD2 protein, and the strongest band represents a post-translationally cleaved ~60 kDa SUSD2 product. (c) IHC analysis of SUSD2 in HGSOC cells/spheroids. HGSOC cell lines and OVCAR3 spheroids were pelleted into buttons before being formalin fixed and embedded in paraffin. OVCAR3 spheroids were formed in 24 h by plating OVCAR3 cells in ultra-low attachment plates (20 000 cells/spheroid). Using an anti-SUSD2 antibody, IHC analysis was performed on HGSOC cell line monolayers and OVCAR3 spheroids. Brown indicates positive SUSD2 staining, and blue indicates cell nuclei (counterstained with hematoxylin). Representative images are shown imaged at x200 magnification. (d, e) Cell proliferation analysis. HGSOC cell lines were plated in triplicate in selection media and counted using a Coulter counter every 24 h for 6 days (144 h) to assess cell growth. (e) HGSOC cell counts over the course of linear growth were used to calculate cell-doubling time.