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[Study of RON mediated invasion of Raji cell line and drug-target effects]
Bi-cui Zhan1, Yue-han Dong, Jian Fan
1The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Objective:
To study the proto-oncogene RON mediated aggression of Raji cells and the inhibitory effects by monoclonal antibody Zt/f2 (2f2).
Methods:
The effects of RON ligand macrophage stimulating protein (MSP) (2.0 nmol/L) and inhibitory Zt/f2 (2F2) (2.0 nmol/L) antibody on proliferation of RON positive Raji cells after treatment for 24 and72 hours were detected by MTT method, colony formation units (CFU) of Raji cells by methylcellulose semi solid culture, Raji cells apoptosis and cell cycle analysis by AnnexinV/PI double staining, expression of RON, apoptosis-related proteins, and cyclins by Western blot.
Results:
(1)Compared with the cell viability (1.0) and counts of CFU (103.6±7.0) in control group, Raji cells after MSP treatment had better viability (1.35±0.20) and CFU counts (133.7±10.4) (P<0.05), but worse viability (0.68±0.11) and CFU counts (66.3±6.1) after Zt/f2 (2F2) treatment (P<0.05). (2)Percentage of Raji cells apoptosis after Zt/f2 (2F2) antibody treatment (12.16±2.33)% was significantly increased than the control (2.89±1.03)% (P<0.05). The percentage of Raji cells arrested in G0/G1 phase was increased after Zt/f2 (2F2) antibody treatment as compared to the control [ (54.96 ±3.70)% vs (39.10±2.30)%, (P<0.05) ]. (3) High-level of RON phosphorylation and β-catenin expression activated by MSP could be inhibited significantly by Zt/f2 (2F2), which also up-regulated the expression of caspase-3, caspase-8, caspase-9 and PARP and down-regulated anti-apoptotic MCL-1 gene and inhibitor of apoptosis protein XIAP expression, accompanied with G1 phase protein changes accordingly.
Conclusion:
MSP could aggravate Raji cells proliferation. Inversely, Zt/f2 (2F2) could inhibit proliferation and induce apoptosis by inhibition of RON phosphorylation and up-regulation of apoptosis related proteins.
Insights
Macrophage stimulating protein (MSP) aggravates Raji cell proliferation, while the monoclonal antibody Zt/f2 (2F2) inhibits proliferation and induces apoptosis by targeting the proto-oncogene RON signaling pathway.
Area of Science:
- Oncology
- Cell Biology
- Immunology
Background:
- The proto-oncogene RON (Recepteur d'Origine Nantais) plays a role in cell proliferation and survival.
- Raji cells, a human B-cell lymphoma line, express RON and are utilized to study its effects.
- Understanding RON-mediated signaling is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the role of proto-oncogene RON in Raji cell aggression.
- To evaluate the inhibitory effects of the monoclonal antibody Zt/f2 (2F2) on RON-mediated Raji cell proliferation and survival.
Main Methods:
- Raji cells were treated with macrophage stimulating protein (MSP) or Zt/f2 (2F2) antibody.
- Cell proliferation was assessed using MTT assays and colony formation units (CFU).
- Apoptosis, cell cycle, and protein expression (RON, apoptosis-related proteins, cyclins) were analyzed via AnnexinV/PI staining and Western blot.
Main Results:
- MSP treatment increased Raji cell viability and CFU counts, indicating enhanced proliferation.
- Zt/f2 (2F2) treatment significantly reduced cell viability and CFU counts, while increasing apoptosis.
- Zt/f2 (2F2) inhibited RON phosphorylation and β-catenin expression, upregulated pro-apoptotic proteins (caspase-3, -8, -9, PARP), and downregulated anti-apoptotic proteins (MCL-1, XIAP), leading to G1 cell cycle arrest.
Conclusions:
- Macrophage stimulating protein (MSP) promotes Raji cell proliferation.
- The monoclonal antibody Zt/f2 (2F2) effectively inhibits Raji cell proliferation and induces apoptosis.
- Zt/f2 (2F2) exerts its effects by inhibiting RON phosphorylation and modulating apoptosis-related protein expression.
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