Gateway cloning system kit

The vector of pENTR-D-Topo-β1 was previously established in our laboratory47 (link). We then used a Gateway^TM cloning System kit (Invitrogen) for getting the expression vectors. Briefly, a LR clonase reaction (Invitrogen) was used to transfer the cDNAs of β1 from the entry vectors into CSII-EF-Rfa. The CSII-EF-β1 was cotransfected with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev into 293T cells. After infection for 48 h, the virus media was collected. The KO cells were infected with the resultant virus for 72 h, and the β1 positive cells were selected using the FACSAria II (BD Biosciences) twice. The stable cell line was used in subsequent studies.

The vector of N-Terminal p3XFLAG-CMV with target cDNA was previously established in our laboratory [18 (link)]. We then used a Gateway^TM cloning System kit (Invitrogen) for getting the expression vectors. The Gateway donor vector RelA from pDONR221 was recombined into Gateway destination vector pCW 57.1 by LR recombination [41 (link)]. The pCW 57.1 (Addgene, #41393) vector alone (Empty vector, Mock) was used as the control. For transient expression, MDA-MB-231 cells were transfected with plasmid constructs (1 μg) by using iMFectin Poly DNA Transfection Reagent (#I7200, GenDEPOT) according to the manufacturer’s instructions.

The cDNA of the human TM4SF1 and integrin α6 were amplified by PCR from the reverse-transcribed product of Eca109 cell total RNA and then subcloned into the pDONR201 vector as described previously [38 (link)]. The resultant cDNAs were confirmed by sequence. The cDNAs of TM4SF1 and integrin α6 from the pDONR201 were transferred into pLenti-CMV-Puro DEST (w118-1) (Addgene, #17452), pLenti-CMV-Hygro DEST (w117-1) (Addgene, #17454), or pLX304 (Addgene, #25890) vectors via LR reaction (GatewayTM cloning system kit, Invitrogen, Thermo Fisher Scientific, Massachusetts, USA).
For expressing short hairpin RNA, we used the pLKO.1-puro lentiviral vector. Inserted oligonucleotide sequences were listed as follows: shRNA#1 against TM4SF1, 5′-CCGGGTTTCCCATTCATACACCTATCTCGAGATAGGTGTATGAATGGGAAACTTTTT-3′, shRNA#2 against TM4SF1, 5′-CCGGTCAAGTAATAAATGGAGTGCTCTCGAGAGCACTCCATTTATTACTTGATTTTT-3′, shRNA#1 against integrin α6, 5′-CCGGGACAACGTGATCCGGAAATATCTCGAGATATTTCCGGATCACGTTGTCTTTTTG-3′, and shRNA#2 against integrin α6, 5’- CCGGTTCTTTAACTGCCGTAATTTACTCGAGTAAATTACGGCAGTTAAAGAATTTTTG-3′.