Figure 2.

Activation of spindle assembly checkpoint in SON-depleted cells. (A) Western blot analysis of lysates derived from HeLa cells pre-treated with two independent SON-specific (SON-1 and SON-2) or control (CTR) siRNAs. (B) SON is required for cell proliferation. Immediately after siRNA transfection, cells were seeded onto separate 6 cm² dishes. At indicated time points cells were harvested and counted using a hemocytometer. (C) SON-depletion results in accumulation of mitotic cells. 36 hr post-transfection, siRNA-treated cells were fixed in 70% ethanol, immunostained with anti-phospho-H3 antibodies and subjected to flow cytometry analysis to evaluate percentage of mitotic cells. (D and E) SON-depletion activates the MAD2-dependent spindle-assembly checkpoint. Western blot showing protein levels in lysates derived from cells pre-treated with indicated siRNAs (D). Percentage of mitotic cell population in siRNA-treated cells (E). Procedures were as described in Methods and in (C). Results represent mean ± S.D from three experiments.