Lentivirus expressing shRNA for pLKO-shZO-1 or pLKO-shCLDN7 were purchased from sigma. To produce lentivirus expressing shRNA co-transfected with the lentivirus packaging plasmids (psPAX2, pMD2G, and VSV-G) into 293T cells. The virus-containing cell culture supernatant was harvested, filtered through a 0.22-μm pore-size filter, and used to infect EC96 cells. To generate stable ZO-1 and CLDN7 knockdown cells, infected cells were selected by puromycin (2 μg/mL), and several rounds of single-cell cloning. The establishment of E-cadherin knockdown EC96 stable cell lines were described previously [27 (link)].
Specific siRNAs, targeting ZO-1, CLDN1 CLDN7, and non-specific control siRNAs were purchased from Santa Cruz Biotechnologies. For transient transfection, cells were seeded at a density of 5 × 104 cells/mL in an antibiotic-free medium, and siRNAs were transfected using the transfection reagent (Santa Cruz Biotechnology, Dallas, TX, USA), according to the manufacturer’s instructions. After incubation for 48 h, the cells were analyzed using in vitro wound-healing assay.
Specific siRNAs, targeting ZO-1, CLDN1 CLDN7, and non-specific control siRNAs were purchased from Santa Cruz Biotechnologies. For transient transfection, cells were seeded at a density of 5 × 104 cells/mL in an antibiotic-free medium, and siRNAs were transfected using the transfection reagent (Santa Cruz Biotechnology, Dallas, TX, USA), according to the manufacturer’s instructions. After incubation for 48 h, the cells were analyzed using in vitro wound-healing assay.
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