Dual luciferase reporter assay system

TET:Rps26 cells were transformed with plasmids encoding firefly luciferase preceded by an 10-adenosine upstream sequence and Renilla luciferase preceded by one of six sequences (listed in Table S1 and Figure S4a). Cells were then grown in selective media, depleted of Rps26 as described above and harvested in mid-log phase. Control experiments demonstrate that the copy numbers of these plasmids do not change upon dox addition (Figure S4c), ensuring that dox-dependent differences we see arise from differential translation, although we cannot exclude effects on mRNA transcription. For luciferase assays under stress, WT cells were grown for 4h under stress conditions (or in minimal media as controls) as detailed below and harvested in mid-log phase. Cells were lysed and luciferase activity was measured using the Promega Dual-Luciferase Reporter Assay System on a Perkin Elmer EnVision 2104 Multilabel Reader according the manufacturer’s protocol with assay volumes scaled down to 15%.

The original HOPFLASH reporter plasmid was from Addgene (#83467, a gift from Barry Gumbiner^{37 (link)}). We subcloned the 8X MCAT from HOPFLASH into a lentiviral backbone. The sequence from human CTGF between −600 to 0 and human FZD7 promoter area between −597 to −123 were amplified by PCR and cloned to generate CTGF and FZD7 luciferase reporter. Human TEAD1 promoter between −650 to +404 were amplified by PCR and then connected by overlap PCR with the left part of 5′UTR and CDS_0–18 which were synthesized by IDT directly to generate T1R. PCR and overlap PCR were performed using primers with MCAT mutation and T1R as template to generate T1MR. Luciferase assay were performed with Dual-Luciferase KIT (Promega) according to the manufacturer’s instructions. Briefly, cells were transfected with luciferase reporters and the target genes for 24 h, and then luciferase (Firefly and Renilla) activity was measured from 20 μL cell lysate with the Dual-luciferase Reporter Assay System on a multilabel plate reader (Wallac Victor, PerkinElmer). The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the Renilla luciferase activity or total protein from the same well. Total protein level were measured by BCA method.