Supplementary Figure 4: Cytohesin activity alters Exportin-5 localization and expression has varying effects on patient outcomes.

a. Control or SecinH3 treated LOX cells were fractionated as detailed in methods. Equal amounts of nuclear and cytosolic fractions were resolved by SDS-PAGE and probed as indicated by western blotting. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. b. Western blotting using 10 μg of total protein from cells treated with vehicle control or SecinH3 show no reduction in total levels of Exportin-5. Representative blots (N=3 biologically independent samples) shown. c. Similar to western blotting, qRT–PCR showed no change in Exportin-5 mRNA levels upon inhibition of the cytohesins by treatment with SecinH3. Data presented as mean±SD for N=3 biologically independent experiments. No significant relationship was found by one-way ANOVA with Tukey’s correction for multiple comparisons. d. LOX cells treated with SecinH3 alone or SecinH3 and Chloroquine were fractionated as outlined in methods. Cytosolic Exportin-5 levels were measured by western blotting of equal amounts of nuclear and cytosolic fractions. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. e. High levels of GRP1 expression correlate with poor overall survival. f. Cytohesin 1 and cytohesin 2 show an inverse correlation between expression levels and overall survival in pan-cancer analysis. Kaplan-Meier data reported with log-rank test statistic (χ²) and the p-value (χ²-distribution). In all panels, P-values ≤0.05 were considered significant. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.