Figure 9.
Rates of RNA export and levels of cytosolic RNA are regulated by available GTP. (A) Representative IF images of BJ-5ta cells treated with EU for 1 h (pulse), then re-fed for 0 or 3 h with EU-free media (chase) either untreated (control) or treated with leptomycin B. (B and C) Quantification of (B) cytoplasmic to nuclear ratio and (C) total EU labeling of BJ-5ta cells treated with leptomycin B pulsed with EU for 1 h, then chased for 3 h (B; control n = 97, leptomycin B n = 92) or 0 h (C; control n = 119, leptomycin B n = 128). Results are from two independent replicates. (D and E) Quantification of (D) cytoplasmic to nuclear ratio or (E) total EU labeling of BJ-5ta cells treated with MPA pulsed with EU for 1 h, then chased for 3 h (D; control n = 178, MPA n = 125) or 0 h (E; control n = 111, MPA n = 125). Each contains two independent replicates. (F) Representative IF images of BJ-5ta cells treated with EU for 1 h (pulse), then re-fed for 0 or 3 h with EU-free media (chase) treated with cycloheximide. (G and H) Quantification of (G) cytoplasmic to nuclear ratio or (H) total EU labeling of BJ-5ta cells treated with cycloheximide and pulsed with EU for 1 h, then chased for 3 h (G; control n = 146, cycloheximide n = 112) or 0 h (H; control n = 103, cycloheximide n = 106). Each contains two independent replicates. (I and J) Quantification of (I) cytoplasmic to nuclear ratio or (J) total EU labeling of BJ-5ta cells depleted of SUN2 pulsed with EU for 1 h, then chased for 3 h (I; siControl n = 175, siSUN2 n = 161) or 0 h (J; siControl n = 92, siSUN2 n = 79). Each contains three independent replicates. (K and L) Quantification of (K) cytoplasmic to nuclear ratio or (L) total EU labeling for cytochalsin B-treated BJ-5ta cells pulsed with EU for 1 h, then chased for 3 h (K; control n = 190, CytoB n = 175) or 0 h (L; control n = 140, CytoB n = 103). Each contains two independent replicates. (M) Representative confocal images of BJ-5ta cells treated with either MPA or leptomycin B labeled with live cell RNA dye for 1 h. (N and O) Quantification of (N) cytoplasmic to nuclear ratio (control n = 582, LeptoB n = 517, MPA n = 409) or (O) total cell fluorescence (control n = 606, LeptoB n = 536, MPA n = 269) across three independent replicates. (P) Representative confocal images of BJ-5ta cells depleted of SUN2 labeled with live cell RNA dye for 1 h. (Q and R) Quantification of (Q) cytoplasmic to nuclear ratios (siControl n = 122, siSUN2 n = 149) or (R) total cell fluorescence (siControl n = 86, siSUN2 n = 86) of live cell RNA dye in cells depleted of SUN2. Each contains three independent replicates. Significance was calculated using unpaired t test (B–E, G–J, Q, and R) or one-way ANOVA with Dunnett’s post hoc (N and O). ns P > 0.05. P****<0.0001. Scale bars 10 µm.

Rates of RNA export and levels of cytosolic RNA are regulated by available GTP. (A) Representative IF images of BJ-5ta cells treated with EU for 1 h (pulse), then re-fed for 0 or 3 h with EU-free media (chase) either untreated (control) or treated with leptomycin B. (B and C) Quantification of (B) cytoplasmic to nuclear ratio and (C) total EU labeling of BJ-5ta cells treated with leptomycin B pulsed with EU for 1 h, then chased for 3 h (B; control n = 97, leptomycin B n = 92) or 0 h (C; control n = 119, leptomycin B n = 128). Results are from two independent replicates. (D and E) Quantification of (D) cytoplasmic to nuclear ratio or (E) total EU labeling of BJ-5ta cells treated with MPA pulsed with EU for 1 h, then chased for 3 h (D; control n = 178, MPA n = 125) or 0 h (E; control n = 111, MPA n = 125). Each contains two independent replicates. (F) Representative IF images of BJ-5ta cells treated with EU for 1 h (pulse), then re-fed for 0 or 3 h with EU-free media (chase) treated with cycloheximide. (G and H) Quantification of (G) cytoplasmic to nuclear ratio or (H) total EU labeling of BJ-5ta cells treated with cycloheximide and pulsed with EU for 1 h, then chased for 3 h (G; control n = 146, cycloheximide n = 112) or 0 h (H; control n = 103, cycloheximide n = 106). Each contains two independent replicates. (I and J) Quantification of (I) cytoplasmic to nuclear ratio or (J) total EU labeling of BJ-5ta cells depleted of SUN2 pulsed with EU for 1 h, then chased for 3 h (I; siControl n = 175, siSUN2 n = 161) or 0 h (J; siControl n = 92, siSUN2 n = 79). Each contains three independent replicates. (K and L) Quantification of (K) cytoplasmic to nuclear ratio or (L) total EU labeling for cytochalsin B-treated BJ-5ta cells pulsed with EU for 1 h, then chased for 3 h (K; control n = 190, CytoB n = 175) or 0 h (L; control n = 140, CytoB n = 103). Each contains two independent replicates. (M) Representative confocal images of BJ-5ta cells treated with either MPA or leptomycin B labeled with live cell RNA dye for 1 h. (N and O) Quantification of (N) cytoplasmic to nuclear ratio (control n = 582, LeptoB n = 517, MPA n = 409) or (O) total cell fluorescence (control n = 606, LeptoB n = 536, MPA n = 269) across three independent replicates. (P) Representative confocal images of BJ-5ta cells depleted of SUN2 labeled with live cell RNA dye for 1 h. (Q and R) Quantification of (Q) cytoplasmic to nuclear ratios (siControl n = 122, siSUN2 n = 149) or (R) total cell fluorescence (siControl n = 86, siSUN2 n = 86) of live cell RNA dye in cells depleted of SUN2. Each contains three independent replicates. Significance was calculated using unpaired t test (B–E, G–J, Q, and R) or one-way ANOVA with Dunnett’s post hoc (N and O). ns P > 0.05. P****<0.0001. Scale bars 10 µm.

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