human ppp2r2b vector  (OriGene)

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 3: PPP2R2B Hypermethylation modulates MYC and P70 phosphorylation to Induce BEZ235 resistance. (A) PPP2R2B CpG island methylation was determined in CNE2 and CNE2/235 cell lines using a pyrosequencing assay. (B) The levels of PPP2R2B mRNA were determined using a quantitative PCR assay in parental and resistant cell lines. (C) The interactions between MYC and PP2A and between P70 and PP2A were detected by immunoprecipitation in CNE2 and CNE2/235 cell lines. (D) The levels of PPP2R2B mRNA were examined by q-PCR after transfection with PPP2R2B shRNA in CNE2 and HONE1. (E) Effect of PPP2R2B knockdown with shRNA on MYC and P70 phosphorylation in CNE2 cell line. (F) Effect of PPP2R2B knockdown on the interactions between MYC and PP2A and between P70 and PP2A in the CNE2 cell line based on immunoprecipitation. (G) The sensitivity of CNE2 to BEZ235 after PPP2R2B knockdown by PPP2R2B shRNA using the MTT assay.

Article Snippet: Plasmid construction, retrovirus production and infection The human PPP2R2B vector was from Origene (RC220271).

Techniques: Phospho-proteomics, Methylation, Pyrosequencing Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, shRNA, Knockdown, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 6: Inhibition of DNA Methyltransferase activity reverses BEZ235 resistance. (A) The mRNA levels of DNA methyltransferases were analyzed in CNE2 and CNE2/235 cell lines by q-PCR. (B) PPP2R2B and PTEN mRNA levels were analyzed in resistant cell lines after treatment with the DNA methylation inhibitor decitabine at the indicated concentrations for 72 h. (C) Effects of decitabine on the expression of survival pathway proteins at the indicated concentrations. (D) Effects of decitabine on the binding of MYC and PP2A and of P70 and PP2A in the CNE2/235 cell line. Cells were treated with 1 μM decitabine for 48 h. Immunoprecipitation was conducted. (E) Effect of the DNA methylation inhibitor decitabine on cell proliferation in the parental and resistant cell lines using the MTT assay. (F) Inhibitory effect of decitabine and BEZ235 on cell proliferation in CNE2 and CNE2/235 cell lines. Cells were treated for 5 d at the indicated concentrations. The MTT assay was conducted. (G) The IC50 values of BEZ235 with or without decitabine were determined in CNE2 and CNE2/235 cell lines using the MTT assay. The data shown are representative of 3 individual experiments.

Article Snippet: Plasmid construction, retrovirus production and infection The human PPP2R2B vector was from Origene (RC220271).

Techniques: Inhibition, Activity Assay, DNA Methylation Assay, Expressing, Binding Assay, Immunoprecipitation, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 7: Targeting methyltransferase overcomes BEZ235 resistance in vivo. (A) (B) Xenograft tumor growth of CNE2 and CNE2/235 cells in nude mice treated with BEZ235 at 25 mg/kg per day or decitabine 1.5 mg/kg three times per week. (p values in CNE2: p = 0.27 in Decitabine vs slovent, p < 0.01 in BEZ235 vs NS, p < 0.01 in combination vs solvent, p = 0.55 in combination vs BEZ235; p values in CNE2/235: p = 0.90 in Decitabine vs slovent, p = 0.03 in BEZ235 vs NS, p < 0.01 in combination vs solvent, p = 0.03 in combination vs Decitabine) (C) The inhibitory rates of tumor growth were calculated as 100% × (1-average treated tumor size/ average control tumor size). (D) Antitumor efficacy of BEZ235 and decitabine in vivo by tumor weight. Nude mice were killed, and tumors were isolated to weigh each tumor. (E) The mRNA levels of PPP2R2B and PTEN were determined by q-PCR in parental and resistant xenografts. (F) Protein expression of survival pathway components was determined by immunoblotting analysis in parental and resistant xenografts. (G) Protein expression of survival pathway components was determined in parental and resistant xenografts by immunoblotting analysis following treatment with decitabine in vivo.

Article Snippet: Plasmid construction, retrovirus production and infection The human PPP2R2B vector was from Origene (RC220271).

Techniques: In Vivo, Solvent, Control, Isolation, Expressing, Western Blot