Plko 1 pur

The methods for vector construction and generation of stable cell lines were described in our previous study [30 (link)]. The oligonucleotides to suppress integrin β₅ expression were designed by RiboBio (Guangzhou, China). After we verified their knockdown efficiency, they were cloned into lentiviral expression vector pLKO.1‐Pur (Addgene, Cambridge, MA, USA). The plasmids were verified by sequencing. Empty vector pLKO.1‐Pur carrying a scrambled shRNA served as a control. 293T cells were incubated with the constructed vectors, pMD2G and psPAX2 (Addgene), according to the manufacturer's protocol. 0.22 μm PVDF filters were used to filter 293T cells supernatant, and then, the supernatant was added into the plate to infect DLD1, HCT116, and HCT8 cells. The oligonucleotide sequences for vector construction are listed in Table S4.

Pfu Ultra II Fusion HS DNA Polymerase (Stratagene, Agilent Technologies) was used to amplify the cDNA encoding RPPH1 or TUBB3 and then the cDNA was cloned into lentiviral expression vector pSin-EF2-Pur reformed from pSin-EF2-Sox2-Pur (Addgene, Cambridge, MA, USA). The oligonucleotides to suppress RPPH1 or TUBB3 expression were designed by RiboBio (Guangzhou, China). Then they were cloned into lentiviral expression vector pLKO.1-Pur (Addgene, Cambridge, MA, USA). The plasmids were verified by sequencing. Empty vector pSin-EF2-Pur and vector pLKO.1-Pur carrying a scrambled shRNA served as a control. 293T cells were incubated with the vectors described above, psPAX2 and pMD2G (Addgene) according to the manufacturer’s instructions. The supernatant containing infectious lentivirus was filtered through 0.22 μm PVDF filters after harvesting at 24 h post transfection and then added into the plate to infect SW620 and HCT8 cells. Infection efficiency was confirmed by qRT-PCR. The methods for transfection and lentiviral infection were described in a previous study^{43 (link)}. The oligonucleotide sequences for vector construction are listed in Supplementary Table 5.