Transfection and Luciferase Assay Protocol

For transfection experiments, MC were plated at 50% confluency and transfected with either 1 µg of the mouse TSP1 luciferase reporter construct [mTSP1-luciferase, a gift from P. Bornstein, Plasmid #12409, Addgene (Michaud-Levesque and Richard, 2009 (link))] with 0.05 µg pCMV β-galactosidase (β-Gal, Clonetech) using Effectene (Qiagen) or 100 nM of MTJ1 or control siRNA (Silencer Select, ThermoFisher) using Lipofectamine (Invitrogen). After 18 h, cells were serum-deprived and treated as above for protein collection for siRNA experiments. For luciferase harvest, 1× Reporter Lysis Buffer (Promega) was added to the plate which was then stored at −80°C overnight prior to cell lysis. Luciferase activity was measured on clarified lysate using the Luciferase Assay System (Promega) with a luminometer (Junior LB 9509, Berthold). Β-Gal activity was used to normalize transfection efficiency, measured using the β-Galactosidase Enzyme Assay System (Promega) with a SpectraMax Plus 384 Microplate Reader (Molecular Devices) set to read absorbance at 420 nm.
The pcDNA3.1 plasmid which contains GRP78 lacking its ER retention sequence KDEL was transfected by electroporation as previously described (Trink et al., 2022 (link)). This was used to overexpress GRP78 at the cell surface. The empty vector pcDNA 3.1 was used as a control.