Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Op18 and MAP4 function as counteractive regulators of monomer-polymer partitioning of tubulin dimers at interphase in the B-cell lymphoma line DG75 and the T-cell leukemia line Jurkat.
  The experiments were performed and data are presented as described for Fig. 1C using DG75 cells (A) or Jurkat cells (B). The shRNAs used for depletion of Op18 and/or MAP4 proteins were the same as in Fig. 1C.
  The results show prominent opposing effects of Op18 and MAP4 in both cell types, which are consistent with the data from K562 cells shown in Fig. 1C. It should be noted that depletion of Op18 and/or MAP4 has no detectable effect on the MT polymer content of mitotic spindles in DG75 and Jurkat cells (data not shown), which agrees with data obtained with K562 cells (see Figs. 1D and S2). Hence, the present findings appear to be general, at least for cells of hematopoetic origin. The data are representative of at least two independent transfection experiments.
• Supplemental Figure 2 - Depletion of Op18 and/or MAP4 has prominent counteractive effects in interphase cells but does not alter the MT polymer content of mitotic spindles significantly.
  Flow cytometric analysis of the distribution of MT-specific fluorescence in the specifically gated interphase population (upper panel) or mitotic population (lower panel) of transfected K562 cells expressing the indicated shRNA, which are described and characterized in Fig. 1B, C and D. Gray graphs show control staining in the absence of anti-α-tubulin but in the presence of fluorescein-conjugated rabbit anti-mouse immunoglobulin.
  Each histogram represents a total of 150,000 cells, and provided the primary data for calculation of the mean values of mitotic cells shown in Fig. 1D. The results do not reveal any detectable phenotypes among mitotic cells (lower panel), while a clear-cut counteractive phenotype is observed among interphase cells (upper panel). This indicates that the opposite effects of Op18 and MAP4 are restricted to the interphase of the cell cycle, which would be consistent with functional phosphorylation-mediated inactivation of both of these MT regulators during mitosis. The data are representative of five independent transfection experiments
• Supplemental Figure 3 - S3. Counteractive phosphorylation-regulation of monomer-polymer partitioning of tubulin dimers during interphase by ectopic MARK2 and CaMKIV(c) kinases in the Burkitt B-cell lymphoma line DG75 and the T-cell leukemia line Jurkat.
  The experiments were performed and data are presented as described for Fig. 2C using DG75 (A) and Jurkat (B) cells. The results show prominent counteractive activities of ectopic MARK2-HA and CaMKIV(c)-F kinases in both cell types, which are consistent with the data from K562 cells shown in Fig. 2C. It should be noted that ectopic MARK2 and CaMKIV(c) kinases had no detectable effect on the MT polymer content of mitotic spindles in DG75 and Jurkat cells (data not shown), which agrees with data obtained with K562 cells (see Fig. 2D). Thus, the present findings on the phenotypes of ectopic protein kinases appear to be general, at least for cells of hematopoetic origin. The data are representative of two independent transfection experiments.