Figure S1. Cytokinetic defects in dsc1... | Rockefeller University Press

Figure S1.

$Cytokinetic defects in dsc1 null mutants generated in the Ax2 background. (A)dsc1 null mutant clones generated in the Ax2 background were verified using Western analysis. Western blot showed absence of DscI protein in all dsc1 null mutant clones. Dynacortin (Dyn) is provided as a loading control. (B) Sequencing analysis confirmed mutations on the dsc1A coding genes of dsc1 mutant clones. Sequences of WT and five separate clones are presented. Nucleotide changes are shown in blue, deletions in red, and insertions in green. (C) Quantification of DscI protein level of the Western blot provided in A. Average integrated intensity of each DscI band was background-subtracted and normalized to their corresponding Dyn control band. Each value was then normalized to WT. (D) Representative growth curves showed that dsc1 null mutants in Ax2 background also display mild growth defects in suspension culture. Cell densities, as indicated, were plotted as a function of time. Growth curve for WT (vector control) cell line is shown in black and those for dsc1 null mutant clones are shown in shades of blue and purple. dsc1 mutant clone 2,4 and 5 were used for this experiment. n = 3 per growth curve, bars represent SEM. (E) Growth rates quantified during exponential growth showed mild yet significant defects in growth in different dsc1 mutant clones. Number of cells analyzed, n = 9–15 from three independent experiments. (F)dsc1 mutants generally stopped growing at a significantly lower saturated density as compared to WT control. For E and F, P values were calculated using ANOVA followed by Fisher’s LSD test. Number of cells analyzed, n = 9–15 from two independent experiments. (G) Quantification of the number of nuclei per cell when cells were 3 d in suspension culture. All dsc1 mutant clones analyzed became more multinucleated compared to WT control. n = 2 per cell line with 100–200 cells per cell line quantified per replicate. (H) Graph shows quantification of fraction of cells detaching when WT and dsc1 cells at equal densities were rotated at different speeds on 6-well non-culture-treated plates. Amount of cells detaching was normalized to the seeding cell number, and the ratio of cell detached over total cell number was determined. P values were calculated using ANOVA followed by Fisher’s LSD test. Number of samples analyzed, n = 3. Source data are available for this figure: SourceData FS1.$

Cytokinetic defects in dsc1 null mutants generated in the Ax2 background. (A) dsc1 null mutant clones generated in the Ax2 background were verified using Western analysis. Western blot showed absence of DscI protein in all dsc1 null mutant clones. Dynacortin (Dyn) is provided as a loading control. (B) Sequencing analysis confirmed mutations on the dsc1A coding genes of dsc1 mutant clones. Sequences of WT and five separate clones are presented. Nucleotide changes are shown in blue, deletions in red, and insertions in green. (C) Quantification of DscI protein level of the Western blot provided in A. Average integrated intensity of each DscI band was background-subtracted and normalized to their corresponding Dyn control band. Each value was then normalized to WT. (D) Representative growth curves showed that dsc1 null mutants in Ax2 background also display mild growth defects in suspension culture. Cell densities, as indicated, were plotted as a function of time. Growth curve for WT (vector control) cell line is shown in black and those for dsc1 null mutant clones are shown in shades of blue and purple. dsc1 mutant clone 2,4 and 5 were used for this experiment. n = 3 per growth curve, bars represent SEM. (E) Growth rates quantified during exponential growth showed mild yet significant defects in growth in different dsc1 mutant clones. Number of cells analyzed, n = 9–15 from three independent experiments. (F)dsc1 mutants generally stopped growing at a significantly lower saturated density as compared to WT control. For E and F, P values were calculated using ANOVA followed by Fisher’s LSD test. Number of cells analyzed, n = 9–15 from two independent experiments. (G) Quantification of the number of nuclei per cell when cells were 3 d in suspension culture. All dsc1 mutant clones analyzed became more multinucleated compared to WT control. n = 2 per cell line with 100–200 cells per cell line quantified per replicate. (H) Graph shows quantification of fraction of cells detaching when WT and dsc1 cells at equal densities were rotated at different speeds on 6-well non-culture-treated plates. Amount of cells detaching was normalized to the seeding cell number, and the ratio of cell detached over total cell number was determined. P values were calculated using ANOVA followed by Fisher’s LSD test. Number of samples analyzed, n = 3. Source data are available for this figure: SourceData FS1.

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