BioMed Research International / 2014 / Article / Fig 5

Research Article

Effects of Crude Extracts from Medicinal Herbs Rhazya stricta and Zingiber officinale on Growth and Proliferation of Human Brain Cancer Cell Line In Vitro

Figure 5

(a) CAERS and/or CFEZO treatment(s) modulated the expression of the NF-κBp65 and p53 proteins. The U251 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for 24 h. Subsequently, 20 μg of whole cell (or nuclear) protein extract was isolated from treated cells and subjected to SDS-PAGE in 10% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with antibodies against the depicted proteins. The immunoblots showed that CAERS and/or CFEZO treatment(s) downregulated expression level of NF-κBp65 protein found in whole cell or nuclear extract but did not alter level of NF-κBp65 found in the cytosolic extract. The immunoblot showing nuclear NF-κBp65 was stripped off and reprobed with anti-PCNA antibody as an internal loading control for nuclear extract. The CAERS and/or CFEZO treatment(s) upregulated expression level of p53 protein. Representative blots from several independent experiments are shown. (b) CAERS and/or CFEZO treatment(s) modulated the expression of the displayed antiapoptotic and proapoptotic gene products. After CAERS and/or CFEZO treatment(s) total RNA was then isolated, reverse-transcribed, and subjected to PCR with gene-specific primers. The PCR products of the genes were then subjected to electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. GAPDH was used as the internal control, M, DNA ladder. The data are representative of three separate experiments.