Figure 6: HyperSUMO conditions initiate tumorsphere formation.

(A) After 72 hr siRNA treatment, MCF10-2A cells were evaluated for viability using a cell counter. All experiments were performed in duplicates. The graph represents the mean ± SEM of 3 independent experiments. (B) To test for propagation/colony formation abilities of normal MCF10-2A cells in the presence and absence of SENP7S, 1000 single cells were grown for 5 days after siNT or siSENP7S treatment and colonies (visible in the accompanying photograph) were counted. Student’s t-test indicates that knockdown of the large SENP7S population in MCF10-2A cells significantly enhances colony formation. (C) Single cell suspension of the appropriate stable SENP7S-deficient (shSENP7S) and non-targeting shRNA (shNT) MCF10-2A cells was generated. Ten thousand cells were grown in nonadherent media conditions for 8 days and imaged. White and yellow bars represent 500 μm and 100 μm, respectively. Tumorspheres highlighted with a black-dashed box were magnified in the insert. Yellow circle illustrates the size difference between shNT versus shSENP7S spheroids. (D) Total number of tumorspheres was counted in 3 random magnification fields for 3 independent experiments. Graph illustrates mean ± SEM and statistical significance using Student’s t-test. (E) First passage tumorspheres were harvested, RNA purified, and subjected to real-time PCR for assessment of the indicated mRNA. The fold-change in the indicated mRNA (2^ΔΔCt values) is shown; raw ΔCt values were used for Student’s t-test analysis of shSENP7S versus shNT tumorspheres. (F,G) Single cell suspensions of first passage tumorspheres were generated and cultured in nonadherent conditions to test for formation of second-generation spheroids. After 10 days, images were taken and the number of tumorspheres was assessed as described above (D).