Coelenterazine substrate

Cultures of human bone marrow-derived MSCs from healthy de-identified adult volunteer donors (31–33 years old, Table S1) were established as previously described (Lennon and Caplan, 2006 (link)) after informed consent. The bone marrow was collected using a procedure reviewed and approved by the University Hospitals of Cleveland Institutional Review Board (IRB# 09-90-195). MSCs from different donors are indicated as donor 3 or 10. Lentiviral constructs for the COL2A1 promoter were placed upstream of the Gaussia luciferase reporter gene. MSCs were transduced with the COL2A1 reporter lentivirus as previously described for a SOX9 reporter (Correa et al., 2018 (link)). MSCs with the COL2A1 luciferase reporter were expanded as indicated above. At different time points during chondrogenesis, medium of MSCs with the COL2A1 reporter was harvested 48 h after the last medium renewal. Per condition, 50 μl of the medium was transferred to a white 96-well plate and 20 μM coelenterazine substrate (NanoLight technology) was injected into the wells. The Gaussia Luciferase (Gluc) activity was measured using a GloMax-96 Microplate Luminometer (Promega) in technical duplicates.

Control and CELF1 KD myoblasts were transfected using Lipofectamine^™ 2000 (Life Technologies) [31 (link)] in a 96-well plate format with two reporter plasmids; one encoding a Firefly luciferase (FLuc; pLightSwitch_3’UTR from SwitchGear Genomics) and the other encoding a Gaussia luciferase (GLuc) bearing an N-terminal signal peptide [33 (link)]. Approximately 18 hr post-transfection, activity of the secreted GLuc in the media was measured by addition of the coelenterazine substrate (Nanolight Technology). Also, to account for variation in transfection efficiency, the FLuc activity in the cells was measured using the Steady Glo reagent (Promega). The signal from each luciferase was read in a Victor plate reader (Perkin Elmer). The GLuc reading was normalized to that of FLuc in each experiment.