Lipojet in vitro transfection kit ver 2

To knock down the endogenous expression of host genes or ectopic viral ORF57 expression in HEK293T cells, 5 ×10⁵ cells seeded in a 6-well plate were transfected with 40 nM SMARTpool human siRNAs (Horizon) or non-targeting (siNT) control siRNA (Horizon) using LipoJet In Vitro Transfection Kit (Ver. II) (SignaGen Laboratories). Twenty-four hours after the first transfection, the cells were divided into two wells and subjected to the second round of siRNA transfection, followed by transfection of ORF57-expression or empty vectors. After 48 h, total RNA extraction was extracted by TriPure reagent for RT-qPCR. Protein lysates obtained by direct cell lysis in 2 × LDS protein sample buffer supplemented with 5% (vol/vol) 2-ME were analyzed by Western blotting for the protein expression of FOS, MTR4, RGS2, AKT, pAKT, GAPDH, and ORF57.

Primary mouse keratinocytes from newborn C57Bl/6NCr mouse were cultivated as described [83 (link)] in the presence of Rho kinase inhibitor Y-27632 (Enzo Life Sciences, #ALX-270-333). SMARTpool human siRNAs targeting Pard3 (#M-040036-01-0005) and Grip1(#M-064716-00-0005) were purchased from Dharmacon. Non-targeting control siRNA (Dharmacon, # D-001210-01) served as a non-specific siRNA negative control. Mouse keratinocytes plated without the Rho kinase inhibitor Y-27632 [84 (link)] were transfected twice with each siRNA (40 nM) at an interval of 24 h using the LipoJet In Vitro Transfection Kit (Ver. II) (SignaGen Laboratories, #SL100468). Total protein and total RNA extracts were prepared 24 h after the second siRNA transfection.