Journal: Nature cancer

Article Title: P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia

doi: 10.1038/s43018-022-00394-x

Figure Lengend Snippet: a) Immunoblot depicting protein levels of phosphorylated PRAS40, FOXO3a, and TSC2 following 24-hour treatment of OCI-AML2 cells with selinexor.

Article Snippet: Membranes were probed with the following primary antibodies (where available: clone, catalogue number, dilution): β-actin (13E5) (CST #4970 diluted 1:5000 in 5% BSA), p-AKT T308 (244F9) (CST #4056 diluted 1:1000 in 5% BSA), p-AKT S473 (D9E) (CST #4060 diluted 1:1000 in 5% BSA), T-AKT (C67E7) (CST #4691 diluted 1:3000 in 5% BSA), p-GSK3β S9 (D85E12) (CST #5558 diluted 1:1000 in 5% BSA), p-BAD S136 (D25H8) (CST #4366 diluted 1:1000 in 5% BSA), XPO1 (C-1) (sc # 74454 diluted 1:100 in 5% BSA), cleaved-PARP (D64E10) (CST #5625 diluted 1:1000 in 5% BSA), cleaved-Caspase3 D175 (CST #9661 diluted 1:500 in 5% BSA), p110-g (D55D5) (CST #5405 diluted 1:1000 in 5% BSA), p110α (C73F8) (CST #4249 diluted 1:1000 in 5% BSA), p110β (C33D4) (CST #3011 diluted 1:1000 in 5% BSA), p110δ (D1Q7R) (CST #34050 diluted 1:1000 in 5% BSA), p101 (D32A5) (CST #5569 diluted 1:1000 in 5% BSA), Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMab ™ Rabbit mAb mix (CST #6967 diluted 1:1000 in 5% BSA), Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit (CST #5569 diluted 1:1000 in 5% BS, T-S6K1 (CST#9202 1:1000 in 5% BSA) or p-S6K1 (CST#9205 1:500 in 5% BSA) overnight (16 hours).

Techniques: Activation Assay, Inhibition, Western Blot

Journal: Nature cancer

Article Title: P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia

doi: 10.1038/s43018-022-00394-x

Figure Lengend Snippet: a) Experimental strategy for parallel assessment of cell-beneficial and cell-detrimental effects of nuclear export inhibition with the XPO1 inhibitor, selinexor. Pooled CRISPR-Cas9 screening in OCI-AML2 cells treated with selinexor reveals genetic modifiers of drug sensitivity. Reverse phase protein array (RPPA) analysis in selinexor treated OCI-AML2 cells reveals drug-responsive protein and phosho-protein expression.

Article Snippet: Membranes were probed with the following primary antibodies (where available: clone, catalogue number, dilution): β-actin (13E5) (CST #4970 diluted 1:5000 in 5% BSA), p-AKT T308 (244F9) (CST #4056 diluted 1:1000 in 5% BSA), p-AKT S473 (D9E) (CST #4060 diluted 1:1000 in 5% BSA), T-AKT (C67E7) (CST #4691 diluted 1:3000 in 5% BSA), p-GSK3β S9 (D85E12) (CST #5558 diluted 1:1000 in 5% BSA), p-BAD S136 (D25H8) (CST #4366 diluted 1:1000 in 5% BSA), XPO1 (C-1) (sc # 74454 diluted 1:100 in 5% BSA), cleaved-PARP (D64E10) (CST #5625 diluted 1:1000 in 5% BSA), cleaved-Caspase3 D175 (CST #9661 diluted 1:500 in 5% BSA), p110-g (D55D5) (CST #5405 diluted 1:1000 in 5% BSA), p110α (C73F8) (CST #4249 diluted 1:1000 in 5% BSA), p110β (C33D4) (CST #3011 diluted 1:1000 in 5% BSA), p110δ (D1Q7R) (CST #34050 diluted 1:1000 in 5% BSA), p101 (D32A5) (CST #5569 diluted 1:1000 in 5% BSA), Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMab ™ Rabbit mAb mix (CST #6967 diluted 1:1000 in 5% BSA), Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit (CST #5569 diluted 1:1000 in 5% BS, T-S6K1 (CST#9202 1:1000 in 5% BSA) or p-S6K1 (CST#9205 1:500 in 5% BSA) overnight (16 hours).

Techniques: Inhibition, CRISPR, Protein Array, Expressing

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: A novel XPO1 inhibitor have anti-esophageal cancer activity and XPO1 is highly expressed in esophageal squamous cancer (A) Cell viability of 21 active compounds against esophageal cancer from clinical compound library. The XPO1 inhibitor verdinexor was identified as an active drug. Cells were seeded in 96-well plates and treated with 20 μM drugs for 48 h and cell viability was measured by CCK-8 assay. (B) XPO1 gene expression from TCGA database and GTEx database analyzed by the GEPIA 2 online tool. (C) Representative western blot analyses XPO1 protein expression in three cases of esophageal carcinoma and normal esophageal tissues. (D) Representative western blot analyses XPO1 protein expression in esophageal squamous cells (KYSE30, KYSE450, KYSE180, KYSE410, and KYSE510) and normal esophageal cells (NE1). The data was analyzed by Student's t-test (two-sided) or one-way ANOVA. Error bars represent ± SD, * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Activity Assay, CCK-8 Assay, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor could bind to XPO1 protein and inhibited cell proliferation in esophageal cancer. (A) Chemical structure of verdinexor. (B) The DARTS assay for target validation. XPO1 protein stability was increased upon KPT-335 (100 μM) treatment in KYSE30 lysates. Pronase treatment was conducted for 20 min. (C) The IC 50 of verdinexor, cisplatin and 5-fluorouracil for 48 h was performed by CCK-8 assay in esophageal squamous cancer cells (KYSE30, KYSE450, KYSE180 and KYSE510). (D) Verdinexor inhibited the colony formation ability of KYSE30 and KYSE450. The data were analyzed by Student's t-test (two-sided) or one-way ANOVA. Error bars represent ± SD, * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor suppressed migration, induced cleavage of PARP and downregulated XPO1 expression in esophageal squamous cancer. KYSE30 and KYSE450 cells were treated with the indicated concentrations of verdinexor for 24 h. Wound width was shown by representative images (A) and the ratio of wound width (B) was analyzed by wound migration assay. Scale bars, 100 µm. (C&D) XPO1, PARP and cleaved PARP, E-cadherin and Vimentin protein expression were detected by western blot assay. The data were analyzed by Student's t-test (two-sided) or one-way ANOVA. Error bars represent ± SD, *P < 0.05, **P < 0.01.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Migration, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor inhibited XPO1/c-Myc/FOSL1 axis. KYSE30 cells were treated with verdinexor for 24 h. (A) Differential genes by RNA-sequence analysis were shown by a volcano map. (B) KEGG pathway enrichment analysis were shown with P < 0.05. (C) Differential genes related with Wnt pathway were shown in a heat map. (D) The interaction of XPO1 and related Wnt pathway differential genes was displayed by PPI network. (E) The FOSL1 gene expression was analyzed by RT-qPCR and protein expression of XPO1, FOSL1 and c-Myc were detected by western blot assay. (F) KYSE30 cells were treated with verdinexor, MG132 or a combination of verdinexor and MG132 for 8 h. FOSL1 and c-Myc expressions were detected by western blot assay. (G) KYSE30 cells were transfected with small RNA of FOSL1 or c-Myc for 24 h and then treated with verdinexor for 24 h. FOSL1, c-Myc and XPO1 expressions were analyzed by western blot assay. The data were analyzed by Student's t-test (two-sided) or one-way ANOVA. Error bars represent ± SD, *P < 0.05, **P < 0.01.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor significantly inhibited nuclear accumulation of c-Myc. KYSE30 cells were treated with verdinexor for 24 h. (A) the colocalization experiment was using immunofluorescence to detect the colocalization of XPO1, c-Myc and FOSL1. Scale bars, 100 µm. (B) Cell fractionation experiments were performed and the expressions of XPO1 and c-Myc in the nucleus and cytoplasm (Cyto) were analyzed by western blot assay.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Immunofluorescence, Cell Fractionation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor disturbed the interaction between XPO1 and c-Myc. KYSE30 cells were treated with verdinexor for 24 h. (A) the interaction of XPO1 and c-Myc was detected by immunoprecipitation analysis and western blot assay. (B) Cell fractionation experiments were conducted and the interaction of XPO1 and c-Myc in the nucleus and cytoplasm was detected by immunoprecipitation analysis and western blot assay. (C) The interaction of XPO1 and c-Myc was detected by proximity ligation assay. Scale bars, 100 µm.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Immunoprecipitation, Western Blot, Cell Fractionation, Proximity Ligation Assay

Journal: International Journal of Biological Sciences

Article Title: Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

doi: 10.7150/ijbs.66612

Figure Lengend Snippet: Verdinexor inhibited tumor growth and suppressed XPO1 and c-Myc expression in vivo . (A) KYSE30 cells were implanted in nude mice, treated with control and verdinexor (5 mg/kg, dissolved in 2 % DMSO, 40 % PEG300, 5 % Tween-80 and 53 % saline). The images showed the relative size of the tumors. Scale bars, 1 cm. (B&C) The tumor volumes were monitored every other day, and once the mice were euthanized, the tumor was weighed immediately. (D) XPO1 and c-Myc expressions were analyzed by immunohistochemistry. Scale bars, 100 µm. The data were analyzed by Student's t-test (two-sided) or one-way ANOVA. Error bars represent ± SD, *P < 0.05, **P < 0.01.

Article Snippet: The antibodies used for PLA were XPO1 (Cell Signaling Technology, #46249, 1:200) and c-Myc (Cell Signaling Technology, #18583, 1:200).

Techniques: Expressing, In Vivo, Immunohistochemistry

Journal: Autophagy

Article Title: Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia

doi: 10.1080/15548627.2019.1707494

Figure Lengend Snippet: Analysis of XPO1 and PPP3/calcineurin in normoxia/hypoxia-exposed primary human trophoblasts and the placenta from e-PE vs. controls. (A–C) The expression of XPO1 and PPP3 in cells with indicated treatment was evaluated using western blotting in A and statistically analyzed in B and C. (D) The phosphatase activity of PPP3/calcineurin was assessed and compared between hypoxia- and normoxia-treated cells. (E–G) Protein abundance of XPO1 and PPP3 was detected using western blotting (E) and statistically analyzed (F and G) in the placenta from e-PE and gestational age-matched deliveries.

Article Snippet: The following rabbit polyclonal Ab were used: anti-TUBB/β-tubulin (Cell Signaling Technology, 2146), anti-KRT7 (Novus Biological, NB120-17069), anti-MAP1LC3B (MBL, PM036), anti-TFEB (Millipore, ABF234), anti-PPP3CA/calcineurin α subunit (Sigma-Aldrich C1956) and anti-XPO1/CRM1 (Cell Signaling Technology).

Techniques: Expressing, Western Blot, Activity Assay