The dual luciferase assays were used to verify the function of the m6A site of PxJHE gene in HKT‐293T cells as described elsewhere.[40 (link)
] The pmirGLO luciferase expression vector (Promega) with a firefly luciferase (F‐luc) and a Renilla luciferase (R‐luc) was used to construct the fluorescent reporter plasmid as described elsewhere.[41 (link)
] Wild‐type PxJHE fragment (PxJHE‐wt) contained CDS and 3′‐UTR of PxJHE and mut‐type PxJHE fragment (PxJHE‐mut) was made by replacing the adenosine bases (A) with cytosine (C). The fragment of PxJHE‐wt and PxJHE‐mut was attached to the pmirGLO vector by In‐Fusion HD Cloning Kit (TaKaRa) according to the instructions. The HKT‐293T cells were seeded in a 24‐well plate using DMEM medium with 10% FBS and 1% antibiotics, and cells were cultured in a CO2 incubator at 37 °C. Transfection experiments were performed when the cell density was ≈ 80%. 200, 400, 600, and 800 ng plasmid of PxJHE‐wt or PxJHE‐mut were transfected into HKT‐293T cells cultured in DMEM medium with 10% FBS using Lipofectamine 3000 (Invitrogen). After 48 h, the cells were lysed, and the fluorescence activity was detected using the Dual‐Glo Luciferase Assay System (Promega). The effect of the m6A site on PxJHE mRNA was evaluated by firefly luciferase activity, and firefly luciferase activity was normalized to Renilla luciferase activity.
] The pmirGLO luciferase expression vector (Promega) with a firefly luciferase (F‐luc) and a Renilla luciferase (R‐luc) was used to construct the fluorescent reporter plasmid as described elsewhere.[41 (link)
] Wild‐type PxJHE fragment (PxJHE‐wt) contained CDS and 3′‐UTR of PxJHE and mut‐type PxJHE fragment (PxJHE‐mut) was made by replacing the adenosine bases (A) with cytosine (C). The fragment of PxJHE‐wt and PxJHE‐mut was attached to the pmirGLO vector by In‐Fusion HD Cloning Kit (TaKaRa) according to the instructions. The HKT‐293T cells were seeded in a 24‐well plate using DMEM medium with 10% FBS and 1% antibiotics, and cells were cultured in a CO2 incubator at 37 °C. Transfection experiments were performed when the cell density was ≈ 80%. 200, 400, 600, and 800 ng plasmid of PxJHE‐wt or PxJHE‐mut were transfected into HKT‐293T cells cultured in DMEM medium with 10% FBS using Lipofectamine 3000 (Invitrogen). After 48 h, the cells were lysed, and the fluorescence activity was detected using the Dual‐Glo Luciferase Assay System (Promega). The effect of the m6A site on PxJHE mRNA was evaluated by firefly luciferase activity, and firefly luciferase activity was normalized to Renilla luciferase activity.
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