Figure 8.
RanGTPase exhibits increased cytoplasmic localization under conditions that elevate levels of available GTP which can be partially reversed by overexpression of NTF2. (A) Western blot analysis of BJ-5ta cells depleted of SUN2 and probed for Ran. Tubulin was used as a loading control. (B) IF images of SUN2-depleted BJ-5ta cells were probed for Ran (green) and stained with Hoechst (blue) to visualize the nucleus. (C) Quantification of cytoplasmic to nuclear ratio of endogenous Ran from BJ-5ta cells treated with siSUN2 (siControl n = 80, siSUN2 n = 69; three independent replicates). (D) Quantification of cytoplasmic to nuclear ratio of endogenous Ran in BJ-5ta cells treated with cytochalasin B (control n = 130, CytoB n = 99; three independent replicates). (E) Representative confocal images of BJ-5ta cells stably expressing GFP-Ran treated with cytochalasin B at indicated time points. Images taken from Videos 1 and 2. (F and G) Quantification and (G) relative rate of change in nuclear to cytoplasmic ratio of GFP-Ran of BJ-5ta cells within the first 2 min of cytochalasin B treatment (control n = 47, CytoB n = 46; three independent replicates). (H) Representative confocal images of GFP-Ran in MCF10A cells treated with cytochalasin B at indicated time points. (I and J) Quantification (I) and relative change (J) in cytoplasmic to nuclear ratio of GFP-Ran in MCF10A cells treated with cytochalasin B (control n = 60, cytoB n = 60; three independent replicates). (K) BJ-5ta cells expressing GFP-Ran were trypsinized and plated on fluorodishes for live cell imaging. Representative graph of the relative levels of GFP-Ran and cellular area over time in a single BJ-5ta GFP-Ran cell. The point at which the cellular area or GFP-Ran localization dramatically changes from initial plating is identified as the time of change (indicated with dashed lines). *In order to combine these measurements on a similar scale, cellular area is represented in pixels and Ran localization change is represented by fluorescence (a.u. × 1,000). (L) Correlation of time of change in GFP-Ran to change in cellular area from (K; n = 18 across two independent experiments). Cells with no change in cellular area were not included in linear regression. (M) Representative IF images of BJ-5ta parental or mCherry-NTF2 (red) overexpressing cells treated with cytochalasin B and probed for Ran (green). (N) Quantification of endogenous cytoplasmic to nuclear Ran from M (parental control n = 85, parental cytoB n = 72, mCherry-NTF2 control n = 77, mCherry-NTF2 cytoB n = 73; three independent replicates). (O) Quantification of endogenous cytoplasmic to nuclear Ran in parental or mCherry-NTF2 expressing BJ-5tas depleted of SUN2 (parental siControl n = 106, parental siSUN2 n = 99, mCherry-NTF2 siControl n = 104, mCherry-NTF2 siSUN2 n = 105; three independent replicates). Significance calculated using t test (C, D, G, and J), one-way ANOVA with Tukey’s post hoc (N and O). ns P > 0.05, P**<0.01, P***<0.001, P****<0.0001. Scale bars 10 µm. Error bars represent ± SEM. Source data are available for this figure: SourceData F8.

RanGTPase exhibits increased cytoplasmic localization under conditions that elevate levels of available GTP which can be partially reversed by overexpression of NTF2. (A) Western blot analysis of BJ-5ta cells depleted of SUN2 and probed for Ran. Tubulin was used as a loading control. (B) IF images of SUN2-depleted BJ-5ta cells were probed for Ran (green) and stained with Hoechst (blue) to visualize the nucleus. (C) Quantification of cytoplasmic to nuclear ratio of endogenous Ran from BJ-5ta cells treated with siSUN2 (siControl n = 80, siSUN2 n = 69; three independent replicates). (D) Quantification of cytoplasmic to nuclear ratio of endogenous Ran in BJ-5ta cells treated with cytochalasin B (control n = 130, CytoB n = 99; three independent replicates). (E) Representative confocal images of BJ-5ta cells stably expressing GFP-Ran treated with cytochalasin B at indicated time points. Images taken from Videos 1 and 2. (F and G) Quantification and (G) relative rate of change in nuclear to cytoplasmic ratio of GFP-Ran of BJ-5ta cells within the first 2 min of cytochalasin B treatment (control n = 47, CytoB n = 46; three independent replicates). (H) Representative confocal images of GFP-Ran in MCF10A cells treated with cytochalasin B at indicated time points. (I and J) Quantification (I) and relative change (J) in cytoplasmic to nuclear ratio of GFP-Ran in MCF10A cells treated with cytochalasin B (control n = 60, cytoB n = 60; three independent replicates). (K) BJ-5ta cells expressing GFP-Ran were trypsinized and plated on fluorodishes for live cell imaging. Representative graph of the relative levels of GFP-Ran and cellular area over time in a single BJ-5ta GFP-Ran cell. The point at which the cellular area or GFP-Ran localization dramatically changes from initial plating is identified as the time of change (indicated with dashed lines). *In order to combine these measurements on a similar scale, cellular area is represented in pixels and Ran localization change is represented by fluorescence (a.u. × 1,000). (L) Correlation of time of change in GFP-Ran to change in cellular area from (K; n = 18 across two independent experiments). Cells with no change in cellular area were not included in linear regression. (M) Representative IF images of BJ-5ta parental or mCherry-NTF2 (red) overexpressing cells treated with cytochalasin B and probed for Ran (green). (N) Quantification of endogenous cytoplasmic to nuclear Ran from M (parental control n = 85, parental cytoB n = 72, mCherry-NTF2 control n = 77, mCherry-NTF2 cytoB n = 73; three independent replicates). (O) Quantification of endogenous cytoplasmic to nuclear Ran in parental or mCherry-NTF2 expressing BJ-5tas depleted of SUN2 (parental siControl n = 106, parental siSUN2 n = 99, mCherry-NTF2 siControl n = 104, mCherry-NTF2 siSUN2 n = 105; three independent replicates). Significance calculated using t test (C, D, G, and J), one-way ANOVA with Tukey’s post hoc (N and O). ns P > 0.05, P**<0.01, P***<0.001, P****<0.0001. Scale bars 10 µm. Error bars represent ± SEM. Source data are available for this figure: SourceData F8.

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