Gv358 lentiviral vector

We first cloned the MAGI2-AS3 DNA sequence into the GV358 lentiviral vector (Genechem, Shanghai, China). We then co-transfected the 293T cells with oeMAGI2-AS3 vector plasmid and helper plasmids for 48 h. We collected the supernatants and purified the lentiviruses carrying the oeMAGI2-AS3 plasmid by centrifugation and further concentrated the virus through ultrafiltration. The ultrapure oeMAGI2-AS3 plasmid and polybrene was incubated with T24 and J82 cells (2 × 10⁵ cell/ml) for 48 h. The efficiency of MAGI2-AS3 overexpression in the BCa cells was confirmed by observing the GFP fluorescence under a fluorescence microscope.

Human CLCA4 cDNA was amplified by PCR and cloned into GV358 lentiviral vector (GeneChem, Shanghai, China). Oligo of CLCA4 shRNAs was synthesized and inserted in GV248 vector Genechem Co., Ltd (GeneChem, Shanghai, China). Stable cell lines that expressed CLCL4 (CLCA4) or CLCA4-shRNAs (CLCA-sh#1 and CLCA-sh#2) were selected for 10 days with 0.5 mg/ml puromycin. In some experiments, cells were treated with LY294002 (50 nM) for 12 h before harvest.

Lentiviral GV248 vector expressing RACK1 shRNA or scramble non-target shRNA, and Lentiviral GV358 vector expressing RACK1 and control vector GV358 were established by Genechem Co. (Shanghai, China), and confirmed by sequencing. The target for human lentiviral shRNA was 5′-CAGGGATGAGACCAACTATGG-3′, the knockdown efficiency of which has been validated^{46 (link)}. SW620 and SW480 cells were infected with the lentiviral particles according to the manufacturer’s instructions, respectively, and then selected using puromycin for 2 weeks. Colon cancer cell lines with stable knockdown or overexpression of RACK1 and control cell lines were obtained.

Lentiviral GV248 vector expressing ANXA1 shRNA or scramble non-target shRNA, and lentiviral GV358 vector expressing ANXA1 were established by Genechem Co. (Shanghai, China), and confirmed by sequencing. The target for human ANXA1 shRNA was 5′-CTTGTATGAAGCAGGAGAA-3′, the knockdown efficiency of which has been validated^{41 (link)}. 5–8F and 6–10B cells were infected with the lentiviral particles respectively, and then selected using puromycin for 2 weeks. NPC cell lines with stable knockdown or overexpression of ANXA1 and control cell lines were obtained.

The DDX17 cDNA (NM_006386.4) fragment was inserted into the lentiviral GV248 vector (Genechem Company). Two short hairpin RNAs (shRNAs) targeting DDX17 (DDX17 shRNA#1: 5′-CAAGGGUACCGCCUAUACC-3′; and DDX17 shRNA#2: 5′-GAGAGACUCUGCAAGCUAU-3′) and negative control (shNC: 5′-UUCUCCGAACGUGUCAGGU-3) were cloned into the lentiviral GV358 vector (Genechem Company), respectively. The packaging and purification of lentivirus were performed by Genechem Company (Shanghai, China). CRC cells with stable DDX17 deficiency or overexpression were selected with puromycin for 1-2 weeks. Human miR-149-3p mimic (5′-AGGGAGGGACGGGGGCUGUGC-3′), miR-149-3p antisense (5′-GCACAGCCCCCGUCCCUCCCU-3′) and CYBRD1 shRNA (5′-UCCAUCCAUGCAGGGUUAAAU-3′) were inserted into the lentiviral LV-3 vector and the lentivirus was packaged by Genepharma Company (Shanghai, China). Small interfering RNAs (siRNAs) targeting CYBRD1 and DDX5 were purchased from Thermo Fisher Scientific. SiRNA was transfected into CRC cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions.