Fig. 4. Effects of overexpression of pub2⁺. (A) h^- wild-type cells (MM72-11C) bearing pREP1-pub2-HA or pREP1-pub2CA-HA were grown up to mid-log phase in EMM2 containing 20 µM thiamine (lane 1, 2) and transferred to thiamine-free EMM2 to overexpress Pub2-HA and Pub2CA-HA (lane 3, 4). Western blotting was conducted with the anti-HA antibody (12CA5). (B) High levels of overexpression of pub2⁺ induces cell elongation. The MM72-11C strain was transformed with plasmid pREP1-pub2-HA (a and c) or pREP1-pub2CA-HA (b and d). Cells were grown up to mid-log phase in EMM2 containing 20 µM thiamine (a and b) and transferred to thiamine-free EMM2 (c and d). After incubation for 18 hours, cell size was observed under phase-contrast optics. Bar, 10 µm. (C) Cell multiplication after thiamine removal. MM72-11C cells transformed with either pREP1 (control) or pREP1-pub2⁺, were grown in EEM2 medium supplemented with 20 µM thiamine to mid-log phase and then transferred to EMM2 without thiamine to induce expression of pub2⁺. pub2⁺-overexpressing cells (closed squares) stopped growing after 16 hours, whereas control cells (open circles) continued to multiply. (D) Flow cytometric analysis for pub2⁺-overexpressing cells. Cells harboring pREP1-pub2⁺ or pREP1 were grown up to mid-log phase in EMM2 containing 20 µM thiamine and transferred to EMM2 without thiamine. Samples were withdrawn at the indicated time points. (E) Accumulation of Cdc25-6HA in pub2⁺-overexpressing cells. The OM1715 strain carrying either pREP1 (an empty vector), pREP1-pub2⁺ or pREP1-pub2CA was incubated in EMM2 without thiamine. Western blotting with anti-HA antibody (12CA5) revealed Cdc25-6HA. Tubulin was detected by anti--tubulin antibody (TAT-1) and was used as an internal reference marker.