LATS Kinase Activity Monitoring

LATS kinase activity was monitored by LATS-BS consisting of two vectors: NLuc-YAP15 (#107610, Addgene) and 14-3-3-CLuc (#107611, Addgene)^{15 (link)}. Lenvatinib-resistant PLC/PRF/5 and Huh7 were transfected with LATS-BS and pRL-CMV Renilla luciferase (Promega) for normalization of transfection efficiency using Lipofectamine 2000. Treatment of 10 µM U0126 or solvent control (DMSO) for 4 hours was done at 48 hour post transfection. The cells were collected and luciferase signal was measured using Dual-luciferase report assay system (#E1910, Promega). Luciferase fold change was calculated according to the method described^{15 (link)}.

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Leung C.O., Yang Y., Leung R.W., So K.K., Guo H.J., Lei M.M., Muliawan G.K., Gao Y., Yu Q.Q., Yun J.P., Ma S., Zhao Q, & Lee T.K. (2023). Broad-spectrum kinome profiling identifies CDK6 upregulation as a driver of lenvatinib resistance in hepatocellular carcinoma. Nature Communications, 14, 6699.

Publication 2023

14-3-3-CLuc pRL-CMV Renilla luciferase Dual-luciferase report assay system

14 3 3 Assay Cells Dmso Kinase Lats Lenvatinib Lipofectamine 2000 Luciferase Promega Renilla luciferase Solvent Transfection U0126 Vectors

Corresponding Organization :

Other organizations : Hong Kong Polytechnic University, Chinese University of Hong Kong, Air Force Medical University, Huazhong University of Science and Technology, Tongji Hospital, Sun Yat-sen University, Sun Yat-sen University Cancer Center, University of Hong Kong

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Variable analysis

independent variables

Treatment of 10 µM U0126

dependent variables

LATS kinase activity, as measured by the LATS-BS consisting of NLuc-YAP15 and 14-3-3-CLuc

control variables

Solvent control (DMSO)
Transfection efficiency, as measured by pRL-CMV Renilla luciferase

controls

Positive control: Not explicitly mentioned.
Negative control: Solvent control (DMSO)

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