Figure 1

SPAG9 expression in breast cancer cells. (a) RT-PCR analysis showed SPAG9 mRNA expression in testis and no expression in normal mammary epithelial cells (NMEC). SPAG9 mRNA expression was observed in MCF-7, MDA-MB-231, BT-474 and SK-BR-3 cells. β-Actin gene expression was used as an internal control. (b) Relative expression of SPAG9 mRNA in MCF7, MDA-MB-231, BT-474 and SK-BR-3 breast cancer cells relative to NMEC. (c) Validation of SPAG9 protein expression in NMEC and breast cancer cells by Western blot analysis. SPAG9 reactive band was detected in MCF-7, MDA-MB-231, BT-474 and SK-BR-3 cell lysates. However, no reactivity against SPAG9 was detected in NMEC. Lower panel depicts the β-actin protein reactivity as an internal loading control in all breast cancer cells. (d) SPAG9 protein expression in breast cancer cells by IIF assay. IIF assay revealed distinct cytoplasmic SPAG9 localization in fixed and permeabilized cells probed with anti-SPAG9 antibody in MCF-7, MDA-MB-231, BT-474 and SK-BR-3 cells. Nuclei of the cells were stained blue with DAPI. All images were captured using confocal microscope (Original magnification, ×630; objective, 63×). (e) SPAG9 surface localization in breast cancer cells. FACS analysis distinctly showed SPAG9 surface localization in MCF-7, MDA-MB-231, BT-474 and SK-BR-3 cells probed with anti-SPAG9 antibody as depicted in histogram plot showing displacement of fluorescence intensity on X axis (M1) as compared to fluorescence intensity of cells stained with secondary antibody only (M2). Representative plots showed high percentages of distinct population of MCF-7 (94.79%), MDA-MB-231 (96.11%), BT-474 (97.39%) and SK-BR-3 (95.21%) cells showing SPAG9 surface localization as compared to cells stained with secondary antibody only.