Low protein binding filter

Single-guide RNA (sgRNA) targeting the predicted ORF for 5′U-HOXC13 were designed with CRISPOR tool (http://crispor.tefor.net/). sgRNA specifically binding the genomic peptide location or upstream with the highest predicted knock out (KO) efficiency were selected. Next, sgRNA were cloned into lenti-Cas9-v2 (RRID:Addgene_52961) with BsmBI.
Lentiviral supernatants were generated by co-transfecting HEK293 cells with lenti-Cas9-v2 encoding the sgRNA of interest, psPAX2 and pMD2G plasmids with PEI (Sigma) and non-supplemented DMEM media (Gibco). Media was replaced after 12 hours with Opti-MEM (Gibco) containing 2% FBS (Gibco). Supernatants were collected at 42 to 48 hours after transfection and filtered through a 0.45-μm low-protein binding filter (Millipore). Patient-derived TCL were infected with lentiviral supernatants containing polybrene (Sigma) final concentration of 8 μg/mL followed by spinoculation 900 g, 32°C, 50 minutes. Five days post-infection, T2 media (RPMI containing 10% FBS supplemented with Pen/Strep and l-Glut) was replaced with T2 media containing puromycin 1 μg/mL to select the cells efficiently transduced. Thereafter, media was replaced with fresh media containing puromycin when acidified or cells split when confluent. To evaluate the KO efficiency, puromycin-resistant cells were used as targets in coculture assays with 5′U-HOXC13 specific T cells.

Lentivirus preparation and transduction were performed according to the manufacturer’s protocol. Lentivirus-based TDO2 small hairpin (sh)RNA (TRCN0000064900), control plasmid (SHC016 1EA), and lentiviral packaging mix were purchased from Sigma-Aldrich. TDO2 lentiviral vector (pLenti-GIII-CMV-Human-TDO2-GFP-2A-Puro Lentiviral Vector, LV332282) was purchased from Applied Biologic Materials (Richmond, Canada). Control, shTDO2, or TDO2 plasmids were cotransfected with the packaging plasmids into 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, the virus-containing supernatants were harvested and centrifuged at 3000 rpm for 15 minutes and filtrated through a 0.45-μm low protein–binding filter (Millipore, Bedford, MA). Hs578T and SUM149 cells were infected with lentivirus (3 multiplicities of infection) in the presence of hexadimethrine bromide (5 μg/mL polybrene; Sigma-Aldrich) and fed with fresh complete medium the next day. Seventy-two hours after infection, the transduced cells were selected by puromycin (2 μg/ml). Efficiency of TDO knockdown or overexpression was validated by qRT-PCR and immunoblot analyses. Cells were cultured thereafter in 1.5 μg/ml puromycin and changed to puromycin-free medium 3–5 days prior to each experiment.

Transfection of the KiSS1 gene into 293T cells was conducted according to the manufacturer’s manual (Origene, USA). Briefly, for transformation, 293T cells were stably packaged with lentviral vector pLenti-C-mGFP-P2A-Puro (Origene) containing KiSS1 DNA (Origene). The lentivirus-containing medium was collected from 293T packaging cell line, the medium was filtrated with 0.22 µm low protein binding filter (Millipore, Germany). At the half-confluent of HepG2 cells then, the lentivirus-containing medium transferred to HepG2 cells (50%-60% confluency) with 10 µg/ml hexadimethrine bromide (Sigma-Aldrich, USA). After infection for 18 h, the lentivirus-containing medium was replaced with fresh cell culture medium and further incubated for 48 h. Transfectants were selected with antibiotics free-medium containing 10 µg/ml puromycin (Sigma-Aldrich) for 48 h, and was repeated 3 times. In this study, the KiSS1-overexpressing HepG2 cell line gene was named HepG2-KiSS1 cells. For vector control group, mock transfection was performed without adding only KiSS1 cDNA clone.