Fig 2: Pharmacologic inhibition of XPO1 with selinexor suppresses neuroblastoma growth in vitro. a. Western blot showing a panel of neuroblastoma cell lines with generally high relative XPO1 expression compared to RPE1. Tubulin was used as loading control for each cell lysate. b. Selinexor dose response curves demonstrate nanomolar range sensitivity across all neuroblastoma cell lines and independent of protein expression. RPE1 is relatively insensitive to selinexor with an IC50 of 41 µM. c. Immunofluorescence of a representative cell line using confocal microscopy shows increased IkB (green GFP-labeled) in the nucleus in response to selinexor. Nuclear staining is shown in blue by DAPI. d. Quantification using Volocity demonstrates the abundance of IkB (GFP signal) in the nuclear (Nuc) compartment was significantly higher with selinexor (SEL) compared to vehicle (VEH) (p-value: < 0.05). No change was observed in the cytoplasmic (Cyto) abundance of IkB in response to selinexor. e. Western blot showing increased IkB in the nuclear compartment (indicated with an arrow) with fractionation using Vinculin as a cytoplasmic marker and Histone H2AX as a nuclear marker. Data shown are representative of 3 experiments with 6 technical replicates (dose response curves) or 3 experimental and technical replicates (IF/Western blots). Densitometry values are representative of 3 independent experiments.

Fig 3: N-terminal phosphorylated Cdh1 is translocated to the cytoplasm by CRM1. a Immunofluorescent staining of shCdh1-MDA-MB-231 cells stably expressing WT-, 4D-, 4A-, Y148E-, or Y148F-Cdh1 using anti-HA(Cdh1) antibody, DAPI was used for DNA staining. *Cdh1 cDNA used in this experiment has been mutated to escape shCdh1-mediated gene silencing. Scale bar, 50 µm. b MDA-MB-231 cells generated in (a) were subjected to cytoplasm/nucleus fractionation followed by immunoblot (IB) analysis. c shCdh1-MDA-MB-231 cells stably expressing the indicated Cdh1 retroviral constructs were subjected to cytoplasm/nucleus fractionation followed by IB analysis. d IB analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) derived from 293T cells transfected with Flag-CRM1 and the indicated HA-Cdh1 constructs. e IB analysis of WCL and anti-HA IP derived from MDA-MB-231 cells generated in (a). f MCF7 and MDA-MB-231 cells were infected with shScr or shCRM1 lentiviral shRNA constructs. The infected cells were selected with 1 µg ml-1 puromycin for 72 h before harvest for cytoplasm/nucleus fractionation and IB analysis. g shCdh1-MDA-MB-231 cells stably expressing WT-, 4D-, Y148E-Cdh1 were infected with shScr or shCRM1 lentiviral shRNA constructs. The infected cells were selected with 1 µg ml-1 puromycin for 72 h before harvest for cytoplasm/nucleus fractionation and IB analysis. h MDA-MB-231 cells were subjected to cytoplasm/nucleus fractionation followed by anti-Cdh1 IP and IB analyses

Fig 4: HOX cluster region shows CRM1-dependent activation of RNA Pol II in LOUCY cells.(A) ChIP-qPCR analysis of active RNA Polymerase II (Pol II) (Ser2- or Ser5-phosphorylated) at HOX gene loci in LOUCY, K562, and HL60 cell lines cultured either in the presence of DMSO (vehicle control) or KPT-330 (1000 nM) for 24 hr. The primer set used was as follows: HOXA4A5 (intergenic region between HOXA4 and HOXA5); HOXA9 (promoter); HOXB4 (promoter); HOXB4 (enhancer); HOXB9 (promoter). Data are presented as mean values ± SEM of four independent experiments (n = 4). (B) The data in (A) were reanalyzed to obtain a ratio by comparing with the value for DMSO treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t-test; **p<0.01. (C–D) Binding profiles of Ser2- or Ser5-phosphorylated Pol II at HOXB (C) or HOXA (D) clusters cultured either in the presence of DMSO (vehicle control) or KPT-330 (1000 nM) for 24 hr. (E) The most affected genes (among CRM1-bound genes) by the treatment with KPT-330. S2P (left panel) and S5P (right panel).

Fig 5: SOD1 binds to BOP1 and WDR12, and regulates the assembly of PeBoW complex. a Flag-SOD1 is prominently localized in the nucleoli. Sod1Flox/Flox KP NSCLC cells stably expressing Flag-SOD1 were treated with 4OHT and doxycycline was added to the medium to induce expression of Flag-SOD1. Cells were fixed on day 5 post treatment and analyzed by immunofluorescence analysis using antibodies against Flag (red) and UBF (green, nucleoli). Dashed lines demarcate the nucleus. Scale bars represent 10 µm. Data are represented by three independent experiments. b Flag-SOD1 is colocalized with BOP1 and WDR12. Sod1Flox/Flox KP cells stably expressing Flag-SOD1 were treated with 4OHT and doxycycline to induce knockout of endogenous Sod1 and expression of ectopic Flag-SOD1, respectively. IF analysis was performed with antibodies against Flag (green) and BOP1 or WDR12 (red). Dashed lines indicate the nucleus. Scale bar represents 10 µm. Data are represented by three independent experiments. c SOD1 interacts with the PeBoW complex in intact cells as judged by Duolink. Sod1Flox/Flox KP cells stably expressing Flag-SOD1 were treated with 4OHT and doxycycline to induce knockout of endogenous Sod1 and expression of ectopic Flag-SOD1, respectively. Cells were fixed and analyzed by Duolink to detect the interaction of Flag-SOD1 and WDR12 or BOP1 (red dots). Shown is a representative image. Scale bars, 10 µm. Data are represented by three independent experiments. d Sod1 knockout or overexpression does not affect the protein level of CRM1, BOP1, and WDR12. Same as (c) except that samples were analyzed by immunoblot. Data are represented by three independent experiments. e Sod1 knockout disrupts the interaction between BOP1 and WDR12 in mouse KP NSCLC cells. Mouse Sod1Flox/Flox KP NSCLC cells were treated with or without 4OHT to knock out endogenous Sod1, and Dox to induced Flag-SOD1 expression. BOP1–WDR12 interaction was analyzed by Duolink. Scale bar represents 10 µm. Data are represented by three independent experiments. f–i SOD1 knockdown disrupts the interaction between BOP1 and WDR12 in human KP and KL NSCLC cells. Human KP and KL NSCLC cells were transfected with SOD1 human siRNA (si-hSOD1) or a control siRNA (siNC). BOP1–WDR12 interaction was analyzed by Duolink. Scale bar represents 10 µm. Data are represented by three independent experiments.