Figure 7.
NuMA and Prc1 co-depletion impact RhoA accumulation at the equatorial membrane. (A–D) IF analysis of HeLa Kyoto cells that are either transfected with control siRNA (A) or siRNA-against NuMA (B), Prc1 (C), or Prc1 and NuMA (DCat.A and DCat.B). Cells were fixed 60 h post siRNA transfection and stained with anti-RhoA (gray) and anti-NuMA (red) antibodies. A minimum of 60 cells from three independent experiments were visually analyzed for RhoA based on their interchromatid distance during anaphase and representative images are shown in the Figure panel. The relative frequency of these category are mentioned in F. Note that NuMA and Prc1 co-depleted cells either show weak (Cat. A) or no RhoA (Cat. B) enrichment at the equatorial membrane. Also, note that Cat. B cells do not show cytokinetic furrow despite significant inter chromatid distance. In Prc1 depleted cells, in addition to its usual membrane localization, NuMA mislocalizes next to the DNA, possibly because of the absence of an intact spindle midzone. The scale bar in the panel and following panels represent 10 μm. (E and F) Schematic representation of the visual quantification of equatorial RhoA membrane intensity (E), and the % of cells based on such visual quantification in various siRNA transfected conditions (F). For such analysis, cells were grouped into four categories: strong (depicted in blue), as in control siRNA transfected cells, reduced (depicted in pink), as in NuMA siRNA, weak (depicted in orange) as in NuMA and Prc1 siRNA Cat. A, and absent (depicted in brown) as in NuMA and Prc1 siRNA Cat. B. (n = 109 cells for control siRNA; n = 81 cells for NuMA siRNA; n = 96 cells for Prc1 siRNA, and n = 60 cells for NuMA and Prc1 siRNA transfected cells from three independent experiments). (G and H) Schematic representation of line scan analysis (G) and the outcome (H) of this analysis for Rho A intensity at the equatorial membrane (depicted in blue) and polar membrane (shown in orange) in different siRNA transfected conditions. Please note a decrease in RhoA equatorial intensity in NuMA siRNA (P < 0.0001) as well as NuMA and Prc1 siRNA (P < 0.0001) transfected cells. Also, note a marginal increase in RhoA intensity at the polar membrane surface in cells transfected with either NuMA siRNA (P = 0.0018) or NuMA and Prc1 siRNA (Cat A: P < 0.04; Cat B: P < 0.0001). The RhoA intensity in Prc1 depleted cells at the polar membrane is non-significant (P = 0.108). n = 15 representative cells from all siRNA conditions were used for this quantification; the shaded region indicates SEM; the intensity value at the peak of each line scan curve has been compared to that of control to measure the P value by two-tailed unpaired Student’s t test. (I and J) Schematic representation of the quantification method (I), and the outcome of such analysis for equatorial membrane enrichment of RhoA fluorescence intensity (J). Please note a significant decrease in equatorial membrane RhoA intensity in cells depleted for NuMA, or NuMA and Prc1. The distribution for the RhoA intensity for all the cells depleted for NuMA and Prc1 is shown in the inset. Note that ∼30% of cells show the ratio of a selected region at the equatorial membrane to that of a similar area at the polar membrane is ∼1 (shown as red dots in the inset). ns, P > 0.05; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 25 cells for control siRNA, NuMA siRNA, or Prc1 siRNA, and n = 27 cells for NuMA and Prc1 siRNA transfected cells were taken from three independent experiments; error bars: SD. (K–R) IF analysis of the central spindle localization of Prc1 (K and O), Cyk4 (L and P), Mklp1 (M and Q), or Ect2 (N and R) in HeLa Kyoto cells that are transfected with either control siRNA or siRNA against Prc1. Yellow-line represents the area that was used for computing the line scan intensity (in the arbitrary unit, au) on the right of each panel. n = 5 cells in each condition, as depicted; error bars: SD. (S) Immunoblot analysis of mitotically synchronized protein extracts made from HeLa Kyoto cells that were either transfected with control siRNA or siRNA against Prc1, NuMA, or Prc1 and NuMA for 60 h. The resulting blot was probed with anti-NuMA, Anti-Prc1, anti-Ect2, anti-Cyk4, and anti-RhoA antibodies. Anti-β-actin antibodies were used to analyze the equal loading of the samples. The values below the Ect2, Cyk4, and RhoA represent the band intensity, normalized to the intensity value from the respective β-actin control. Please note that we have run various % of SDS-PAGE to detect proteins with distinct molecular weight (MW). For instance, NuMA (MW ∼238kD) is detected by running a 6% gel, and for RhoA (MW ∼22 kD) detection, we have run the samples on 12% gel. Source data are available for this figure: SourceData F7.

NuMA and Prc1 co-depletion impact RhoA accumulation at the equatorial membrane. (A–D) IF analysis of HeLa Kyoto cells that are either transfected with control siRNA (A) or siRNA-against NuMA (B), Prc1 (C), or Prc1 and NuMA (DCat.A and DCat.B). Cells were fixed 60 h post siRNA transfection and stained with anti-RhoA (gray) and anti-NuMA (red) antibodies. A minimum of 60 cells from three independent experiments were visually analyzed for RhoA based on their interchromatid distance during anaphase and representative images are shown in the Figure panel. The relative frequency of these category are mentioned in F. Note that NuMA and Prc1 co-depleted cells either show weak (Cat. A) or no RhoA (Cat. B) enrichment at the equatorial membrane. Also, note that Cat. B cells do not show cytokinetic furrow despite significant inter chromatid distance. In Prc1 depleted cells, in addition to its usual membrane localization, NuMA mislocalizes next to the DNA, possibly because of the absence of an intact spindle midzone. The scale bar in the panel and following panels represent 10 μm. (E and F) Schematic representation of the visual quantification of equatorial RhoA membrane intensity (E), and the % of cells based on such visual quantification in various siRNA transfected conditions (F). For such analysis, cells were grouped into four categories: strong (depicted in blue), as in control siRNA transfected cells, reduced (depicted in pink), as in NuMA siRNA, weak (depicted in orange) as in NuMA and Prc1 siRNA Cat. A, and absent (depicted in brown) as in NuMA and Prc1 siRNA Cat. B. (n = 109 cells for control siRNA; n = 81 cells for NuMA siRNA; n = 96 cells for Prc1 siRNA, and n = 60 cells for NuMA and Prc1 siRNA transfected cells from three independent experiments). (G and H) Schematic representation of line scan analysis (G) and the outcome (H) of this analysis for Rho A intensity at the equatorial membrane (depicted in blue) and polar membrane (shown in orange) in different siRNA transfected conditions. Please note a decrease in RhoA equatorial intensity in NuMA siRNA (P < 0.0001) as well as NuMA and Prc1 siRNA (P < 0.0001) transfected cells. Also, note a marginal increase in RhoA intensity at the polar membrane surface in cells transfected with either NuMA siRNA (P = 0.0018) or NuMA and Prc1 siRNA (Cat A: P < 0.04; Cat B: P < 0.0001). The RhoA intensity in Prc1 depleted cells at the polar membrane is non-significant (P = 0.108). n = 15 representative cells from all siRNA conditions were used for this quantification; the shaded region indicates SEM; the intensity value at the peak of each line scan curve has been compared to that of control to measure the P value by two-tailed unpaired Student’s t test. (I and J) Schematic representation of the quantification method (I), and the outcome of such analysis for equatorial membrane enrichment of RhoA fluorescence intensity (J). Please note a significant decrease in equatorial membrane RhoA intensity in cells depleted for NuMA, or NuMA and Prc1. The distribution for the RhoA intensity for all the cells depleted for NuMA and Prc1 is shown in the inset. Note that ∼30% of cells show the ratio of a selected region at the equatorial membrane to that of a similar area at the polar membrane is ∼1 (shown as red dots in the inset). ns, P > 0.05; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 25 cells for control siRNA, NuMA siRNA, or Prc1 siRNA, and n = 27 cells for NuMA and Prc1 siRNA transfected cells were taken from three independent experiments; error bars: SD. (K–R) IF analysis of the central spindle localization of Prc1 (K and O), Cyk4 (L and P), Mklp1 (M and Q), or Ect2 (N and R) in HeLa Kyoto cells that are transfected with either control siRNA or siRNA against Prc1. Yellow-line represents the area that was used for computing the line scan intensity (in the arbitrary unit, au) on the right of each panel. n = 5 cells in each condition, as depicted; error bars: SD. (S) Immunoblot analysis of mitotically synchronized protein extracts made from HeLa Kyoto cells that were either transfected with control siRNA or siRNA against Prc1, NuMA, or Prc1 and NuMA for 60 h. The resulting blot was probed with anti-NuMA, Anti-Prc1, anti-Ect2, anti-Cyk4, and anti-RhoA antibodies. Anti-β-actin antibodies were used to analyze the equal loading of the samples. The values below the Ect2, Cyk4, and RhoA represent the band intensity, normalized to the intensity value from the respective β-actin control. Please note that we have run various % of SDS-PAGE to detect proteins with distinct molecular weight (MW). For instance, NuMA (MW ∼238kD) is detected by running a 6% gel, and for RhoA (MW ∼22 kD) detection, we have run the samples on 12% gel. Source data are available for this figure: SourceData F7.

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