Figure 1. KIRC-2 and -3 express a... | Rockefeller University Press

Figure 1.

$Figure 1. KIRC-2 and -3 express a spontaneous Eg5 rigor mutant, Eg5-G268V. (A) Each KIRC line is clonally distinct. Unsupervised cluster and heatmap analysis of indicated cell types. The dendrogram (bottom) shows that KIRC-1 and KIRC-2 are most similar based on gene expression. HeLa is more similar to KIRC-1 and KIRC-2 than KIRC-3. KIRC-3 is the most distinct cell line. (B) Eg5 binds spindle MTs in KIRC-2 and -3 cells despite the presence of STLC. Single optical sections of indicated cell types stained with antibodies targeting Eg5 (grayscale and red) and tubulin (green). DNA, blue. Lookup tables (LUTs) for grayscale and red channel are scaled identically. Bar, 10 µm. See also Fig. S1. (C) Purification of recombinant Eg5 proteins. (left) Protein gel of Eg5-WT and Eg5-G268V preparations stained with Coomassie-blue. (right) Western blot of Eg5-WT and Eg5-G268V preparations probed with antibodies targeting Eg5 (αEg5). (D) Eg5-G268V is immotile. Kymographs of single XR-MTs in gliding assays propelled by Eg5-WT or Eg5-G268V. Bars: (x axis) 1 µm; (y axis) 1 min. See also Videos 1 and 2. (E) Histogram showing gliding velocities from D. Values represent the number of MTs observed to move at indicated velocities in gliding assays powered by Eg5-WT (green) or Eg5-G268V (red). Velocities are reported in micrometers per minute and binned every 0.1 µm/min. n = 300 from three independent experiments. Distributions of Eg5-WT velocities are fit to a single Gaussian (R2 = 0.91). (F) Eg5-G268V does not release from MTs in the presence of ATP. Blot of pellet (P) and supernatant (S) fractions from reactions containing Eg5-WT or Eg5-G268V with tubulin or microtubules in the presence of DMSO or FCPT as indicated, probed with antibodies targeting Eg5 (αEg5) and tubulin (DM1α).$

KIRC-2 and -3 express a spontaneous Eg5 rigor mutant, Eg5-G268V. (A) Each KIRC line is clonally distinct. Unsupervised cluster and heatmap analysis of indicated cell types. The dendrogram (bottom) shows that KIRC-1 and KIRC-2 are most similar based on gene expression. HeLa is more similar to KIRC-1 and KIRC-2 than KIRC-3. KIRC-3 is the most distinct cell line. (B) Eg5 binds spindle MTs in KIRC-2 and -3 cells despite the presence of STLC. Single optical sections of indicated cell types stained with antibodies targeting Eg5 (grayscale and red) and tubulin (green). DNA, blue. Lookup tables (LUTs) for grayscale and red channel are scaled identically. Bar, 10 µm. See also Fig. S1. (C) Purification of recombinant Eg5 proteins. (left) Protein gel of Eg5-WT and Eg5-G268V preparations stained with Coomassie-blue. (right) Western blot of Eg5-WT and Eg5-G268V preparations probed with antibodies targeting Eg5 (αEg5). (D) Eg5-G268V is immotile. Kymographs of single XR-MTs in gliding assays propelled by Eg5-WT or Eg5-G268V. Bars: (x axis) 1 µm; (y axis) 1 min. See also Videos 1 and 2. (E) Histogram showing gliding velocities from D. Values represent the number of MTs observed to move at indicated velocities in gliding assays powered by Eg5-WT (green) or Eg5-G268V (red). Velocities are reported in micrometers per minute and binned every 0.1 µm/min. n = 300 from three independent experiments. Distributions of Eg5-WT velocities are fit to a single Gaussian (R² = 0.91). (F) Eg5-G268V does not release from MTs in the presence of ATP. Blot of pellet (P) and supernatant (S) fractions from reactions containing Eg5-WT or Eg5-G268V with tubulin or microtubules in the presence of DMSO or FCPT as indicated, probed with antibodies targeting Eg5 (αEg5) and tubulin (DM1α).