Journal: PLoS ONE

Article Title: GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0025634

Figure Lengend Snippet: DNA and proteins were extracted from LNCaP cells treated with increasing doses of 5-aza-CdR (0.005–2.5 µM). Cells were treated daily and DNA and protein harvested after 6 days of treatment. (A) MSP was performed on bisulfite-modified DNA with primers targeting bisulfite-modified methylated GSTP1 promoter or unmethylated GSTP1 promoter. (B–C) the relative methylation status of the GSTP1 promoter following 5-aza-CdR treatment was further assessed by COBRA using two restriction enzymes, BstUI and HhaI. MDA-MB-231 breast cancer cells were used as a control for unmethylated GSTP1 promoter. (D) Immunoblot was performed to analyze GSTP1 protein expression. Detection of Hsp90 was used as a loading control.

Article Snippet: GSTP1 Methylation-Specific Polymerase chain reaction (MSP) and COmbined Bisulfite Restriction Analysis (COBRA) primers were purchased from GeneWorks (South Australia, Australia).

Techniques: Modification, Methylation, Combined Bisulfite Restriction Analysis Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0025634

Figure Lengend Snippet: DNA and proteins were extracted from LNCaP cells after 8 days of treatment with increasing doses of Zebularine (0–400 µM). (A) DNA was bisulfite-modified and MSP was performed with primers targeting bisulfite-modified methylated GSTP1 or unmethylated GSTP1 . (B) The relative DNA methylation status of the GSTP1 promoter following Zebularine treatment was assessed by COBRA using two restriction enzymes, BstUI or HhaI. (C) Immunoblot was performed to analyse GSTP1 protein expression in LNCaP cells after 8 days of Zebularine treatment. Proteins were extracted from LNCaP cells treated with increasing doses of Zebularine (0–400 µM). PC3 cells express endogenous GSTP1 protein and were used as positive control.

Article Snippet: GSTP1 Methylation-Specific Polymerase chain reaction (MSP) and COmbined Bisulfite Restriction Analysis (COBRA) primers were purchased from GeneWorks (South Australia, Australia).

Techniques: Modification, Methylation, DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Western Blot, Expressing, Positive Control