Journal: BMB Reports

Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

doi: 10.5483/BMBRep.2013.46.2.202

Figure Lengend Snippet: Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P ＜ 0.05; **P ＜ 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P ＜ 0.05; **P ＜ 0.01, compared with the untreated control cells.

Article Snippet: The TaqMan TM fluorogenic probes and PCR primers for BRCA1 and gapdh were designed by Metabion International AG (Martinsried, Germany).

Techniques: Expressing, Western Blot, Control, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: BMB Reports

Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

doi: 10.5483/BMBRep.2013.46.2.202

Figure Lengend Snippet: Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P ＜ 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

Article Snippet: The TaqMan TM fluorogenic probes and PCR primers for BRCA1 and gapdh were designed by Metabion International AG (Martinsried, Germany).

Techniques: Expressing, Isolation, Northern Blot, Labeling, Control, Western Blot, Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay, Knockdown