Stable and Transient NQO1 Knockdown/Overexpression

Stable NQO1 knockdown was generated as previously published^{19 (link)}. Briefly, lentiviral particles expressing shRNA against non-targeted control (NTC) and NQO1 (shNQ) were purchased from Dharmacon (Lafayette, CO). LNCaP and PC3 cells were transduced with either NTC or shNQ in the presence of 8 μg/ml polybrene (Sigma-Aldrich) in 96-well plate. Transduction was continued for 48 h. Cells were transferred into a 24-well plate and selected with 1 μg/ml puromycin (Sigma-Aldrich). Serially diluted cells were grown and efficiency of transduction was confirmed in isolated clones by detecting GFP fluorescence. For transient knockdown, cells were transfected with either scramble or NQO1 SMARTpool siRNA (Dharmacon) using Lipofectamine (Invitrogen). Knockdown was verified by qPCR and immunoblot analysis. For stable overexpression, cells were transfected with pcDNA3.1 vector control or pcDNA-NQO1 (pcNQ). After selection with G418, NQO1 overexpression was verified by western blotting.

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Thapa D., Huang S.B., Muñoz A.R., Yang X., Bedolla R.G., Hung C.N., Chen C.L., Huang T.H., Liss M.A., Reddick R.L., Miyamoto H., Kumar A.P, & Ghosh R. (2020). Attenuation of NAD[P]H:quinone oxidoreductase 1 aggravates prostate cancer and tumor cell plasticity through enhanced TGFβ signaling. Communications Biology, 3, 12.

Publication 2020

lentiviral particles polybrene puromycin SMARTpool siRNA Lipofectamine

Cells Clones Fluorescence G418 Immunoblot analysis Lipofectamine Nqo1 Pc3 cells Polybrene Puromycin Shrna Sirna Transient

Corresponding Organization :

Other organizations : The University of Texas Health Science Center at San Antonio, University of Rochester Medical Center

Top 5 similar protocols

Variable analysis

independent variables

Knockdown of NQO1 using shRNA (shNQ)
Overexpression of NQO1 using pcDNA-NQO1 (pcNQ)
Transient knockdown of NQO1 using siRNA

dependent variables

Cell transduction efficiency (confirmed by GFP fluorescence)

control variables

Non-targeted control shRNA (NTC)
Scramble siRNA
PcDNA3.1 vector control

controls

Positive control: Overexpression of NQO1 using pcDNA-NQO1 (pcNQ)
Negative controls: Non-targeted control shRNA (NTC), Scramble siRNA, pcDNA3.1 vector control

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