Journal: Oncology Reports

Article Title: Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-κB and Bcl-2 family in gastric cancer cells

doi: 10.3892/or_00000911

Figure Lengend Snippet: Figure 1. RON siRNA reduces RON expression and cell survival. MKN28 cells were transfected with siRNAs against RON for 3 and 6 days, and the mRNA and protein levels of RON were determined by RT-PCR (A) and Western blot analysis (B). (C) Cells were transfected with scrambled (S) or RON siRNA (si) for 3 and 6 days, and cell viability was determined by an MTT assay. The number of viable cells in the well is expressed as mean ± SD.

Article Snippet: Gene silencing was performed using scrambled (sc-37007) and human RON sequence-specific duplex siRNA (sc-36434; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, MTT Assay

Journal: Oncology Reports

Article Title: Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-κB and Bcl-2 family in gastric cancer cells

doi: 10.3892/or_00000911

Figure Lengend Snippet: Figure 2. The induction of apoptosis by RON siRNA. (A) MKN28 cells were transfected with RON siRNA for 3 and 6 days, and fixed in paraformaldehyde. The DNA was stained using bis-benzimide (Hoechst 33342) to show the presence of nuclear fragmentation and chromatin condensation. (B) Flow cytometric histograms of cells transfected with RON siRNA for 3 and 6 days. The vertical axes (counts) represent the number of cells and the horizonal axes (Annexin-Y FITC) represent the labeling with annexin V. (C) The cells transfected with RON siRNA for 3 and 6 days. The level of the DNA fragmentation was analyzed by electrophoresis in a 1.5% agarose gel.

Article Snippet: Gene silencing was performed using scrambled (sc-37007) and human RON sequence-specific duplex siRNA (sc-36434; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Staining, Labeling, Electrophoresis, Agarose Gel Electrophoresis

Journal: Oncology Reports

Article Title: Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-κB and Bcl-2 family in gastric cancer cells

doi: 10.3892/or_00000911

Figure Lengend Snippet: Figure 3. The effects of RON siRNA on the expression of the Bcl-2 family and the activity of NF-κB. (A) After the transfection of the MKN28 cells with RON siRNA for 3 and 6 days, Western blot analyses were performed with the cell lysates using anti-Bax and Bcl-2 antibodies. (B) Nuclear extracts from the cells were analyzed by EMSA for the activated NF-κB using a radiolabeled oligonucleotide probe. (C) RON siRNA-induced a decrease in the level of the p65 subunit of NF-κB. Protein (50 μg) from the above nuclear lysates were subjected to SDS-PAGE and Western blotting using the NF-κB (p65 subunit) antibody.

Article Snippet: Gene silencing was performed using scrambled (sc-37007) and human RON sequence-specific duplex siRNA (sc-36434; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Activity Assay, Transfection, Western Blot, SDS Page

Journal: Oncology Reports

Article Title: Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-κB and Bcl-2 family in gastric cancer cells

doi: 10.3892/or_00000911

Figure Lengend Snippet: Figure 4. RON siRNA causes the cytochrome c release and caspase activation in cells. (A) After transfection of the MKN28 cells with RON siRNA for 3 and 6 days, Western blot analysis was performed with the cell lysates using the anti-cytochrome c antibody. (B) Caspase-8 and -9 activities were determined using a specific fluorogenic peptide substrate as described in Materials and methods.

Article Snippet: Gene silencing was performed using scrambled (sc-37007) and human RON sequence-specific duplex siRNA (sc-36434; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Crizotinib Has Preclinical Efficacy in Philadelphia-Negative Myeloproliferative Neoplasms.

doi: 10.1158/1078-0432.CCR-22-1763

Figure Lengend Snippet: Figure 4: RON kinase is an important target of crizotinib in MPN (A) Western analysis of RON in SET2 cells treated with crizotinib and stimulated with erythropoietin (EPO), stem cell factor (SCF), and hepatocyte growth factor (HGF). Representative data from one of at least three independent experiments is shown (B) qPCR of MST1R (RON) expression in HEL cells transduced with 4 different MST1R shRNA constructs (C) Colony formation assay of transduced HEL cells in methylcellulose with myeloid growth factors. Experiments were performed at least three times, with similar results. ANOVA with Tukey’s multiple-comparison test was used. (D) Western analysis of RON, STAT3 and STAT5 phosphorylation in shRNA-transduced HEL cells stimulated with EPO, SCF, and HGF. Representative data from one of three independent experiments are shown. (E) Western analysis of JAK2 in shRNA-transduced HEL cells stimulated with EPO, SCF, and HGF (F) Flow cytometry analysis of RON phosphorylation in MPN patient or control donor peripheral blood CD45lo population with ex vivo EPO and SCF stimulation (G) Schematic for JAK2-V617F-GFP retroviral bone marrow transplantation using bone marrow from Stk−/− (RON−/−) mice (H) Peripheral blood analysis of WT (N=8) or Stk−/− (N=9) JAK2-V617F mice over time. WBC= white blood cells; Hb=hemoglobin; PLT=platelets (I) Percentage of GFP in the peripheral blood over time in WT (N=8) or

Article Snippet: Lentiviral Production: Human shRNA plasmid kits for RON (Cat # TL320425) and MET (Cat #TL320418) were purchased from Origene.

Techniques: Western Blot, Expressing, Transduction, shRNA, Construct, Colony Assay, Comparison, Phospho-proteomics, Flow Cytometry, Control, Ex Vivo, Retroviral, Transplantation Assay