Overexpression of JMJD6 in MCF-7 Cells

MCF-7 cell line was transfected with 5μg of vectors expressing JMJD6 WT (pcDNA3-JMJD6WT-V5), JMJD6 mut (pCDNA3-JMJD6MUT-V5) or with empty pCDNA3 (2 X 10⁶ cells in a 100mm dish) using Exgen 500 (Euromedex) according to the manufacturer’s guidelines. Selection was initiated 48h after transfection in medium containing 750μg/ml of G418. After selection, cells were maintained in medium containing 300μg/ml of G418. Three commercially available lentiviral short hairpin RNA (shRNA) constructs targeting human JMJD6 and one negative control construct created in the same vector backbone (pLKO.1-Puro) were purchased from SIGMA. Puromycin selection (1μg/mL) was started 48h after lentiviral infection. The established cell lines have been already described and characterized [23 (link)].

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Poulard C., Rambaud J., Lavergne E., Jacquemetton J., Renoir J.M., Trédan O., Chabaud S., Treilleux I., Corbo L, & Romancer M.L. (2015). Role of JMJD6 in Breast Tumourigenesis. PLoS ONE, 10(5), e0126181.

Publication 2015

Exgen 500 lentiviral short hairpin RNA (shRNA) constructs pLKO.1-Puro

Backbone Cell lines Cells Dish G418 Human Infection Mcf 7 cell Puromycin Shrna Transfection Vector

Corresponding Organization : Institut Gustave Roussy

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Variable analysis

independent variables

Transfection of MCF-7 cell line with 5μg of vectors expressing JMJD6 WT (pcDNA3-JMJD6WT-V5), JMJD6 mut (pCDNA3-JMJD6MUT-V5) or with empty pCDNA3

dependent variables

Not explicitly mentioned

control variables

2 X 10^6 cells in a 100mm dish
Use of Exgen 500 (Euromedex) transfection reagent according to manufacturer's guidelines
Selection initiated 48h after transfection in medium containing 750μg/ml of G418
Cells maintained in medium containing 300μg/ml of G418 after selection
Lentiviral short hairpin RNA (shRNA) constructs targeting human JMJD6 and one negative control construct created in the same vector backbone (pLKO.1-Puro) from SIGMA
Puromycin selection (1μg/mL) started 48h after lentiviral infection

controls

Negative control: Empty pCDNA3 vector
Negative control: Lentiviral shRNA negative control construct

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