Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Inhibiting ERK1/2 activity with CI-1040 does not affect the timing of the G2/M and M/A transitions in PtK and RPE cultures. (A) Western blots reveal that a 15-minute treatment with 100 nM CI-1040 inhibited ERK1/2 activity in PtK and RPE cultures. (B) Cells treated with CI-1040, just as chromosome condensation becomes evident, completed late G2 (prophase) and mitosis without a delay. See also Table 1. Bar = 10 μm.
• Supplemental Figure 2 - Long-term inhibition of ERK1/2 activity does not impede normal bipolar spindle assembly in PTK₁ or RPE1 cells. (A) PtK1 and (B) RPE1 cultures were treated with 50 μM U0126 and fixed after 2, 6, 12 and 24 hrs. They were then immunostained using antibodies against microtubules (α-tubulin; red) and centrosomes (γ-tubulin; green) followed by counterstaining in Hoechst 33342 for the DNA (blue). These images are representative of the prometaphase and metaphase cells found in the cultures after the various time intervals. Cells entering mitosis even after 12-24 hrs in the ERK1/2 inhibitor fored normal bipolar prometaphase and metaphase spindles. There were no mitotic cells in RPE1 cultures after 24 hrs of U0126 treatment. Bar = 10 μm.
• Supplemental Figure 3 - Enhancing ERK1/2 activity with TPA does not prolong late G2 or mitosis in RPE cells. (A) Western blot demonstrating that treatment with 50 or 100 nM TPA significantly enhanced ERK1/2 activity in RPE cultures (for treatment details, see Materials and Methods). B) Cells in which TPA was used to enhance ERK1/2 activity completed late G2 and mitosis with normal kinetics. See also Table 3. Bar = 10 μm.
• Supplemental Figure 4 - Microinjected wildtype his-tagged ERK2 does not localize to centrosomes and centromeres. (A) Kinase assays of purified his-ERK2 was performed using 2 μg of Elk1 protein, a known substrate of ERK2, and 200 μM ATP mixed either with microinjection buffer (Control), 1 ng of control active ERK2 (middle lane) or 1 ng of purified his-ERK2. ERK activity was examined by Western blotting using an antibody against phosphorylated Elk1 (Ser 383). The purified his-ERK2 showed similar kinase activity to that of control ERK2 (supplied with the assay kit). (B) His-tagged wild-type ERK2 protein was microinjected to RPE cells in mid-prophase. After 20-30 min the cells were fixed and immunostained with anti-His antibody. Note that microinjected ERK2 was distributed throughout the cytoplasm, but not on centrosomes or centromeres. Images were selected sections from a z-series. Bar = 10 μm.
• Supplemental Figure 5 - The level of pERK immunostaining associated with chromosomes, centromeres and centrosomes is not noticeably different after knocking ERK 1/2 down with siRNA. (A). Western blots showing that the levels of both kinases (ERK1/2), and their phosphorylated forms (pERK1/2), were significantly decreased 48 hrs after transfection of siRNAs to ERK 1 and ERK2. Blotting for α-tubulin was used as a loading control. (B,C). pERK immunostaining (red) in control mitotic BJ-ELB fibroblasts was concentrated in the centrosomes and chromosome area (blue), often (but not always) colocalizing (yellow) with CREST positive (green) centromere areas (0 hr). Note that this pERK immunostaining distribution is retained even after ERK 1/2 knockdown (right). (D) ERK1/2 knockdown does not interfere with entry into or progression through mitosis (see also Table 1). Mitotic cells were identified in cultures of BJ-ELB fibroblasts, fixed and stained with Hoechst 33342, either after no treatment (white bars) or 48 hrs after transfection of ERK1/2 siRNAs (grey bars). Numbers represent cells in different phases of mitosis as percent of the total number of cells analyzed P = prophase, PM = prometaphase, A = anaphase. Control: 2300 cells, N (Experiments) = 2; ERK1/2 siRNA: 2900 cells, N (Experiments) = 2.