Fig. 7. Extracellular ST6GAL1 regulates cancer stem cell transcription factors in breast tumor cells.

A BT-549 and 4T1.2 cells, as indicated, transfected with the validated shST6GAL1 or the control shRNA were treated with the respective self-exosome particles or in combination with the rST6G for 48 h in a serum-free medium. B A separate culture of shControl and shST6GAL1 4T1.2 cells was used. Validated knockdown cells were treated with self-exosomes or in combination with rST6G, as indicated above. Cells extracts were used for Western blot analysis with the indicated antibodies. CHC, Tubulin, Actin, or GAPDH were used for equal loading transfer. Knockdown of ST6GAL1 in BT-549 and 4T1.2 cells was confirmed by Western blot of ST6GAL1, left panels of A and B, respectively. Ns in A (left panels) highlights a single non-specific band. Experiments were repeated at least three times; representative blots were shown. C, D Human breast cancer T47D cells transfected with the validated shST6GAL1 or the control shRNA were treated with the rST6G or combined with the self-exosome particles (shControl exosomes to shControl cells and shST6GAL1 exosomes to shST6GAL1 cells) for 48 h in serum-free medium. Cell cultures were used for SYBR-Green qPCR analysis with the indicated gene primers. Gene levels were normalized with GAPH and calculated by the delta-delta-Ct method. Data are means ± s.e., n = 3, ANOVA, post-hoc test, *p < 0.05, **p < 0.01 and ***p < 0.001. D Separate cultures of T47D cells were used for Western blot analysis with the indicated antibodies, including ST6GAL1 to validate the knockdown efficiency in shST6GAL1 versus shControl. Tubulin or CHC was used for equal loading and transfer. Experiments were repeated at least three times, and representative blots were shown.