Turner biosystems luminometer

Cell viability was assayed using RealTime-Glo assay (Promega). Briefly, cells were transfected for 24 h and then reseeded at 2 × 10³ cells/well in 96-well plates. MT Cell Viability Substrate and NanoLuc Enzyme were diluted and added to each well. The luminescence was measured with a Turner BioSystems luminometer (Promega) at 24, 48 and 72 h.

Apoptosis was assessed in whole‐kidney lysates using the Caspase‐Glo 3/7 luminescence assay (Promega) measuring levels of active‐caspase 3/7, as described.29 Luminescence was measured using a Turner Biosystems luminometer (Promega, Southampton, UK). Experiments were performed in 10 independent kidney samples each, from RCALC2 mutant mice and WT littermates. Data was expressed as fold change ± SE for each group. Statistical analyses were performed using two‐way ANOVA.

SH-SY5Y cells were co-transfected with the Firefly Luciferase (Fluc) reporter constructs and control Renilla Luciferase plasmid (pRLTK, Promega) and cultured under proliferation or differentiation conditions, as described previously [28 (link)]. Luciferase activity was determined after 24 hours, using the Dual-Luciferase Reporter Assay System (Promega) and Turner Biosystems luminometer (Promega) according to the supplier’s protocol. The levels of Firefly luciferase reporter mRNA were determined using semi-quantitative PCR. No significant differences in stability of the Fluc-Con or Fluc-MBE mRNAs were detected under the conditions employed.

NIH3T3 cells (ATCC CRL-1658)) were co-transfected with the indicated pmiRGLO 3′ UTR reporter plasmid along with either wild-type murine MSI1-eGFP, MSI1-bm-eGFP (which has three phenylalanine to leucine mutations within the first RNA recognition motif (RRM1, F63L/F65L/F68L) that attenuates target RNA association^{37 (link)}), MSI1-AA-eGFP (which is mutated to substitute the two sites of regulatory serine phosphorylation to non-phosphorylatable alanine residues), or eGFP (peGFP N1 empty vector control) plasmids as described previously ^{20 (link),33 (link),34 (link),38 (link),50 (link)}. Expression of the MSI1-eGFP, MSI1-bm-eGFP, and eGFP proteins was confirmed by fluorescence microscopy. Luciferase activity was determined in quadruplicate after 24 h, using the Dual-Luciferase Reporter Assay System (Promega, E2920) and Turner Biosystems luminometer (Promega) according to the supplier's protocol. Data are expressed as relative luciferase activity (FLuc/RLuc) in arbitrary units. All experiments were repeated on at least 3 separate occasions.