xpo1 crm1 nm 003400 expression vector  (OriGene)

Journal: International Journal of Molecular Sciences

Article Title: Nuclear Export Inhibitor KPT-8602 Synergizes with PARP Inhibitors in Escalating Apoptosis in Castration Resistant Cancer Cells

doi: 10.3390/ijms22136676

Figure Lengend Snippet: Schematic diagram illustrating the working hypothesis for the sensitization of mCRPC cells to XPO1 and PARP inhibitors. ( A ) Mechanism of drug resistance with traditional PARP inhibitor and AR-targeted agent treatment. ( B ) Sensitization of mCRPC cells using second generation SINE compound KPT-8602 and PARP inhibitors. BRCA1/2 deficient mCRPC growth cannot be sufficiently blocked by PARP inhibitors and AR blockers since the tumor suppressor proteins (TSPs) are functionally inactivated due to their excessive export to the cytoplasm. KPT-8602 can retain TSPs such as TP53, RB, FOXO, P27 and AR translation promoter eIF4e in the nucleus to facilitate the induction of cell death signals. Moreover, by keeping AR and ARv7 in the nucleus, KPT-8602 interrupts AR- and ARv7-mediated import of ARv1 and ARv6 in the nucleus.

Article Snippet: The mammalian XPO1 (CRM1) (NM_003400) expression vector (pCMV6-CRM1; human-tagged ORF clone) (catalog No. RC206004) and empty vector (pCMV6-Entry) (catalog No. PS100001) were purchased from OriGene (Rockville, MD, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Nuclear Export Inhibitor KPT-8602 Synergizes with PARP Inhibitors in Escalating Apoptosis in Castration Resistant Cancer Cells

doi: 10.3390/ijms22136676

Figure Lengend Snippet: List of primers and sequences used for RT-qPCR.

Article Snippet: The mammalian XPO1 (CRM1) (NM_003400) expression vector (pCMV6-CRM1; human-tagged ORF clone) (catalog No. RC206004) and empty vector (pCMV6-Entry) (catalog No. PS100001) were purchased from OriGene (Rockville, MD, USA).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5

doi: 10.1074/jbc.m602556200

Figure Lengend Snippet: FIGURE 3. Identification of a CRM1-responsive NES in OREBP/TonEBP. A, effectofLMB(10ng/ml)onthesubcellularlocalizationoftheindicatedOREBP mutants expressed in HeLa cells. Representative fluorescence images of fixed cells expressing FLAG-OREBP1–581 and live cells expressing OREBP1–581-GFP. For isotonic treatment, cells transfected with the indicated expression plas- mids were cultured in isotonic medium with or without LMB for 5 h. For hypo- tonic treatment, cells were pretreated with hypertonic medium for 90 min to induce nuclear translocation of the fusion proteins with or without LMB. Sub- sequently, cells were cultured in hypotonic medium for another 90 min with or without LMB. FLAG-OREBP1–581 recombinant protein was visualized after fixation using a FLAG antibody and a FITC-labeled secondary antibody and wasanalyzedbyfluorescencemicroscopy.OREBP1–581-GFPrecombinantpro- tein was visualized by green fluorescence microscopy in live cells. B, align- ment of OREBP/TonEBP nuclear export signal with several characterized NES motifs. The conserved residues are highlighted. C, amino acid sequences of the OREBP/TonEBP NES and illustration of the point mutant. The mutated residues are in boldface. D, functional analysis of the NES. Representative flu- orescence images of fixed HeLa cells. Cells expressing the indicated mutants were treated with hypotonic or isotonic medium for 90 min. Recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody and was analyzed by fluorescence microscopy. Images were examined with 40 objective. Scale bar, 100 m.

Article Snippet: CRM1 cDNA (OriGene Technologies, Inc.) was cloned in-frame into Myc tag expression vector pCMV-Tag-3C (Stratagene).

Techniques: Fluorescence, Expressing, Transfection, Cell Culture, Translocation Assay, Recombinant, Labeling, Microscopy, Mutagenesis, Functional Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5

doi: 10.1074/jbc.m602556200

Figure Lengend Snippet: FIGURE 5. In vivo interaction of NES with CRM1. A, BiFC analysis of interaction between CRM1 and OREBP/TonEBP. Representative fluorescence images of HeLa cells co-transfected with and expressing the indicated plasmids. At 16 h after transfection, cells were treated with hypertonic medium for 2 h and fixed. Fluorescence emission of the cells was imaged. B, quantitation of BiFC signal. The median of yellow fluorescent protein (YFP)/CFP ratios in each group was derivedfrommorethan100transfectedcells.C,expressionofVN173andVC155fusionproteinsasdeterminedbyWesternblotting.HeLacellsweretransfected with plasmids encoding pHA-OREBP1–581VC and pHA-OREBP1–581L4AVC and pHA-CRM1-VN, respectively. Cell lysates were loaded for the determination of protein expression using immunoblotting analysis with anti-HA (for pHA-CRM1-VN), anti-OREBP (for OREBP1–581VC and OREBP1–581L4AVC), and anti-tubulin antibodies. D, effect of overexpressed CRM1 on subcellular localization of OREBP/TonEBP mutants. Myc-CRM1 expression plasmid was transfected into HeLa cells along with expression plasmids for FLAG-tagged OREBP/TonEBP mutants. Cells were treated with hypertonic medium for 2 h, fixed, and stained with anti-FLAG antibody to determine subcellular localization of OREBP/TonEBP mutant proteins (green) and anti-Myc antibody to detect recombinant CRM1 by indirect immunofluorescence microscopy (red). The cells were counterstained with DAPI to visualize nuclei. Images were examined with 40 objective. Scale bar, 100 m.

Article Snippet: CRM1 cDNA (OriGene Technologies, Inc.) was cloned in-frame into Myc tag expression vector pCMV-Tag-3C (Stratagene).

Techniques: In Vivo, Fluorescence, Transfection, Expressing, Quantitation Assay, Western Blot, Plasmid Preparation, Staining, Mutagenesis, Recombinant, Immunofluorescence, Microscopy