Passive cell lysis buffer

EndoC-βH1 cells were seeded at a density of 50,000 per well in 48-well plates. After 48 hours, 0.4 μg of luciferase constructs containing putative regulatory sequences were co-transfected with 1 ng of pRL-Renilla construct as internal control into EndoC-βH1 cells, using Lipofectamine 2000, according to manufacturer’s instruction. pGL3-promoter vector was served as a negative control. 48 h later, transfected cells were washed once with PBS and lysed directly in passive cell lysis buffer (Promega). Cells were incubated on a rotating platform at room temperature for 10 min. to ensure complete lysis of cells, and then spun at 10,000 rpm for 10 min to remove cell debris. Supernatant was transferred into a fresh tube and used to measure luciferase activity with Dual-Luciferase Reporter Assay kit (Promega) on a Lumat LB9507 luminometer (Berthold Technologies). Firefly luciferase measurements were normalized to Renilla luciferase.

EndoC-βH3 cells were seeded at a density of 50,000 per well in 96-well plates. After 48 hours, 0.1 μg of luciferase constructs containing putative regulatory sequences were co-transfected with 1 ng of pRL-Renilla construct as internal control into EndoC-βH3 cells, using Lipofectamine 2000, according to manufacturer’s instruction. pGL4.23 empty vector was served as a control. 48 h later, transfected cells were washed once with PBS and lysed directly in passive cell lysis buffer (Promega). Cells were incubated on a rotating platform at room temperature for 10 min. to ensure complete lysis of cells, and then spun at 10,000 rpm for 10 min to remove cell debris. Supernatant was transferred into a fresh tube and used to measure luciferase activity with Dual-Luciferase Reporter Assay kit (Promega) on a Lumat LB9507 luminometer (Berthold Technologies). Firefly luciferase measurements were normalized to Renilla luciferase.

miRNA-3′-UTR binding sites were identified by microRNA.org. pMIR-REPORT™ System (Life Technologies Co.) was used for reporter assay according to manufacturer instructions and co-transfected with Renilla luciferase vectors, phRL-TK (Promega, Madison, WI, USA), as an internal transfection control. Reporter plasmids were created by cloning putative miR-885-5p target sites in the CPEB2 3′-UTR into pMIR-REPORTER vector downstream of the firefly luciferase gene in pMIR-REPORTER vector. Westermann's lab identified miR-885-5p target sites in the CDK2 and MCM5 were used as positive control with modifications of restriction enzyme: SpeI-3′UTR target site-MluI [30 (link)].
Cells were plated in 24-well plates, and transfected with 200ng of either pMIR empty, pMIR-CPEB2, pMIR-CDK2 or pMIR-MCM5 using Lipofectamine^® 2000 (Life Technologies Co.), or miRNA mimics. After 24-h post transfection, cells were lysed in passive cell lysis buffer (Promega). Lysates were transferred into 96-well plates and processed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer instructions. The luminescence was measured in a FluorStar Optima Microplate Luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity. Transfections were done in triplicate and repeated in three individual experiments.

HEK293T cells were transfected with Renilla luciferase vector pGL4.74 hRL-TK (Promega) or plasmids coding for Renilla luciferase-fused PRRSV-N or nsp1α. Transfected cells were lysed 48 h later using passive cell lysis buffer (Promega) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were further clarified by centrifugation at 13000 rpm for 5 min to remove cell debris then supernatants were transferred to fresh tubes and stored at − 80 °C before use in the LACA assay. Meanwhile, Western blot analysis and an immunofluorescence assay (IFA) were used to test expression levels of recombinant fusion proteins.
Luciferase levels within cell lysates were evaluated using Renilla luciferase substrate (Transgene Biotech, Beijing, China) according to the manufacturer’s instructions and quantified using a VICTORX™ Multilabel Reader (Perkin-Elmer Life and Analytical Sciences, Wellesley, MA, USA). Recombinant Renilla luciferase (Raybiotech, Norcross, GA, USA) was used to determine the relative enzymatic activities of luciferase-fused PRRSV-N or nsp1α from cell lysates. For the LACA, enzyme activity values were assigned to HEK293T cell lysate dilutions in phosphate buffered saline (PBS) (100 μL) based on one enzyme activity equivalent of 1 ng recombinant Renilla luciferase defined as 1 Test Unit (1 TU) unless otherwise specified.

Luciferase assays were carried out 24 h post-transfection unless otherwise indicated. The cells were washed once with PBS and lysed using 0.1 mL of passive cell lysis buffer (Promega, Madison, MI, USA; E1941). After 10 min incubation on a rocker plate, 5 µL sample of the lysed cells was mixed with 50 µL of Luciferase Assay Reagent (Promega; E1501) dispensed in 5 mL polystyrene luminometer tubes (Sarstedt; 55.476.305). Light output was measured using a Sirius luminometer (Berthold, Oak Ridge, TN, USA) programmed to perform a 2-s measurement delay followed by a 10-s measurement read for luciferase activity. Each transfection was done in triplicate wells, with 3–6 individual biological replicates.