Figure 4. Cells tolerate... | Rockefeller University Press

Figure 4.

$Cells tolerate super-stoichiometric α-tubulin. (A) Representative Western blot of α- and β-tubulin during TUB1 induction. Blots were probed for α- or β-tubulin and Zwf1 (G6PD) as a loading control. (B) Quantification of TUB1 induction for both α- and β-tubulin across overexpression. Data from 24 h post-induction is from a separate set of experiments. Dots represent mean ± SD from four independent induction experiments, open circle indicates the separate experiment used to measure tubulin levels at 24 h post-induction. (C) Quantification of cells forming colonies after TUB1 or empty vector induction. Dots represent mean ± SD from three independent induction experiments. (D) Example images of wild-type cells overexpressing TUB1 for 0 or 2 h and stained for α- or β-tubulin. Arrows indicate astral microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of in focus z positions. (E) Quantification of astral microtubules across induction time for both α- and β-tubulin staining. Dots represent mean ±95% CI from three independent experiments with at least 100 cells per experiment. (F) Quantification of tubulin assemblies across induction time for both α- and β-tubulin staining. Dots represent mean ±95% CI from three independent experiments with at least 100 cells per experiment. (G) Example images of cells expressing the indicated + TIP and mRuby-Tub1 with TUB1 induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (H) Quantification of the fraction of cells exhibiting a focus of +TIP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ±95% CI. Source data are available for this figure: SourceData F4.$

Cells tolerate super-stoichiometric α-tubulin. (A) Representative Western blot of α- and β-tubulin during TUB1 induction. Blots were probed for α- or β-tubulin and Zwf1 (G6PD) as a loading control. (B) Quantification of TUB1 induction for both α- and β-tubulin across overexpression. Data from 24 h post-induction is from a separate set of experiments. Dots represent mean ± SD from four independent induction experiments, open circle indicates the separate experiment used to measure tubulin levels at 24 h post-induction. (C) Quantification of cells forming colonies after TUB1 or empty vector induction. Dots represent mean ± SD from three independent induction experiments. (D) Example images of wild-type cells overexpressing TUB1 for 0 or 2 h and stained for α- or β-tubulin. Arrows indicate astral microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of in focus z positions. (E) Quantification of astral microtubules across induction time for both α- and β-tubulin staining. Dots represent mean ±95% CI from three independent experiments with at least 100 cells per experiment. (F) Quantification of tubulin assemblies across induction time for both α- and β-tubulin staining. Dots represent mean ±95% CI from three independent experiments with at least 100 cells per experiment. (G) Example images of cells expressing the indicated + TIP and mRuby-Tub1 with TUB1 induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (H) Quantification of the fraction of cells exhibiting a focus of +TIP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ±95% CI. Source data are available for this figure: SourceData F4.