Shgfp

The human acute monocytic leukemia cell line THP-1 (ATCC: TIB-202^TM) and human LUAD cell lines HCC827 (ATCC: CRL-2868^TM) and A549 (ATCC: CCL-185^TM) were acquired from American Type Culture Collection (ATCC). THP-1 and HCC827 were cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA), and A549 was cultured in DMEM/F12 (Gibco, Carlsbad, CA, USA). All cell lines were cultured in a medium supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin solution, and maintained at 37 °C with 5% CO₂. These three cell lines were confirmed to be mycoplasma free and were passaged < 10 times after the initial recovery of the frozen stocks. All cell lines were authenticated by performing short tandem repeat profiling before use.
Human TIAM2 complement DNA (cDNA) expression vector was constructed by Public Protein/Plasmid Library (Nanjing, China) with pLVX-EF1alpha-IRES-Puro (catalog no. 631988; Clontech, Mountain View, CA, USA).
The shRNA vectors specifically targeting TIAM2 (shTIAM2#1: 5′-GTTAAGGTGATTCGTTCTATT-3′; shTIAM2#2: 5′-GCCCTACTAAAGACATCGAAA-3′) and a scramble control vector (shGFP: 5′-GCAAGCTGACCCTGAAGTTCA-3′) were purchased from Genechem (Shanghai, China). All the plasmid vectors were verified by performing sequencing [84 (link)].

Human hepatocytes (L02) and hepatic tumor cell lines (SK-Hep-1, HepG2, Hep3B, SMMC-7721, Huh-7, Bel-7402 and Li-7) were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), where they were characterized by DNA fingerprinting and isozyme detection. All the cell lines were revived every 3 to 4 months. SK-Hep-1, HepG2, Hep3B, SMMC-7721 and Huh-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). Bel-7402, Li-7 and L02 cells were cultured in RPMI-1640 medium with 10% FBS. All the cell lines were grown at 37 °C in a 5% CO₂/95% air atmosphere.
Four lentiviral shRNAs targeting CBX8 (shCBX8) were synthesized on a GV248-puro vector (Genechem, Shanghai, China). Vector expressed GFP alone was used as a negative control (shGFP, provided by Genechem). According to the manufacturers’ instructions, CBX8 shRNAs were transfected into SK-Hep-1 cells using Lipofectamine 2000 reagent and detected after cultured for 2 to 3 days. Finally, shCBX8-1# (target sequence GGACGTGACCTCAAACTTT) and shCBX8-3# (target sequence TCGCTTGCTCGCAGCCTTT) were chosen for follow-up assays.