Lentiviral Vector Construction and Transfection

Both 5-aza (Decitabine) and MK2206 2HCl were purchased from Selleck Chemicals (Shanghai, China). The skeleton plasmid of lentiviral vectors Flag-vIL-6, His-vIL-6, Flag-STAT3, and Flag-STAT3-K685R was pHAGE-CMV-MCS-IzsGreen (pHAGE for short). pHAGE contains ZsGreen fluorescent coding sequence, which can display fluorescence similar to that produced by green fluorescent proteins in cells. All the shRNAs were constructed into the modified pCDH (named as mpCDH), a new lentiviral plasmid generated in our previous study [32 (link)]. HEK293T cells were cotransfected with lentiviral plasmids, packaging plasmid psPAX2, and pseudotyping pMD2.G using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) as previously described [10 (link), 55 (link), 56 (link)].

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Li W., Wang Q., Qi X., Guo Y., Lu H., Chen Y., Lu Z., Yan Q., Zhu X., Jung J.U., Tosato G., Gao S.J, & Lu C. (2020). Viral interleukin-6 encoded by an oncogenic virus promotes angiogenesis and cellular transformation by enhancing STAT3-mediated epigenetic silencing of caveolin 1. Oncogene, 39(23), 4603-4618.

Publication 2020

5-aza (Decitabine) MK2206 2HCl Lipofectamine 2000 Reagent

5 aza Cells Coding sequence Decitabine Fluorescence Green fluorescent proteins Lipofectamine 2000 Mk2206 Phage Plasmid Shrnas Skeleton Stat3 Vectors

Corresponding Organization : Nanjing University of Chinese Medicine

Other organizations : Nanjing Medical University, Jiangsu Province Hospital, University of Southern California, National Cancer Institute, Center for Cancer Research, UPMC Hillman Cancer Center

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Variable analysis

independent variables

5-aza (Decitabine)
MK2206 2HCl

dependent variables

Fluorescence (similar to green fluorescent proteins) in cells

control variables

Skeleton plasmid of lentiviral vectors Flag-vIL-6, His-vIL-6, Flag-STAT3, and Flag-STAT3-K685R (pHAGE-CMV-MCS-IzsGreen)
ShRNAs constructed into the modified pCDH (mpCDH) lentiviral plasmid
Packaging plasmid psPAX2
Pseudotyping plasmid pMD2.G
HEK293T cells

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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