Regulation of P21 Gene Promoter Activity

The P21 gene promoter sequence from −2000 to +100 was inserted into the reporter plasmid containing firefly luciferase. The specific sequence can be found in the

supplementary document

. The PCR method was used to amplify this sequence, and then it was cloned into the pPRO-RB-REPORT vector (RIOBIO, Guangzhou, China) containing the firefly luciferase wild-type reporter gene. Based on previous research, we chose U-CH1 for dual-luciferase reporter gene experiment.24 (link) The vector was then transfected into U-CH1 cells with or without the si- GSK-3β vector. After transfection for 48 h, the cells were harvested, and luciferase activity was measured using the dual-luciferase reporter gene detection system (Promega, Madison, WI, USA), and normalized for Renilla activity.

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Chen L., Zuo Y., Pan R., Ye Z., Wei K., Xia S., Li W., Tan J, & Xia X. (2021). GSK-3β Regulates the Expression of P21 to Promote the Progression of Chordoma. Cancer Management and Research, 13, 201-214.

Publication 2021

pPRO-RB-REPORT vector dual-luciferase reporter gene detection system

Cells Firefly luciferase Gene Gene promoter Gene system Luciferase Plasmid Promega Renilla Reporter gene Transfection Vector

Corresponding Organization :

Other organizations : Guilin Medical University, Guangxi Zhuang Autonomous Region Brain Hospital

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Variable analysis

independent variables

Presence or absence of si-GSK-3β vector

dependent variables

Luciferase activity

control variables

Renilla activity (used for normalization)

controls

Positive control: U-CH1 cells transfected with the reporter plasmid containing the P21 gene promoter sequence but without the si-GSK-3β vector
Negative control: Not explicitly mentioned

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