Luciferase activities

The reporter vectors including the wild-type of circBRWD3(circBRWD3-WT) and RAC1 (RAC1-WT) as well as the mutant of circBRWD3 (circBRWD3-Mut) and RAC1 (RAC1-Mut) were cloned into pGL3-basic vector (Genechem, China), which were co-transfected with miR-142-3p mimics or miR-142-5p mimics into BC cell lines for 72 h. The luciferase activities (Promega, USA) were then calculated by a Luciferase Reporter Assay System (Ashland, USA).

The gene sequence of the SerRS promoter was amplified by the polymerase chain reaction (PCR) from the HEK 293 cell line using a forward primer: 5′-TCAGGTACC AGTAGAGACGGGATTTTGTCATG-3′ and reverse primer: 5′-TCACTCGAGCAAATCCAGATCCAGCACCATCTTC-3′ with Kpn I and Xho I restriction enzyme sites. The fragment was then subcloned into the pGL4.11 [luc2P] vector. A stably-transfected MDA-MB-231 cell line containing a SerRS promoter-driven firefly luciferase [pGL4.11 (luc2P)] and Renilla (pRL-SV40) was constructed for the purpose of screening for molecules that activated the promoter of SerRS. Stably-transfected MDA-MB-231 cells were seeded into 24-well plates, incubated for 24 h, exposed to experimental compounds or DMSO as a control for 48 h, and the luciferase activities measured (Promega, Madison, WI, USA).

To investigate NF-κB activation, the dual luciferase approach was adopted to detect the NF-κB promoter activity. Briefly, HEK293T cells were grown in 24-well cell culture plates at 60–80% confluency. For parasite treatment, pNF-κB-Luc plasmids containing firefly luciferase gene under the control of NF-κB promoter and pRL-TK, which acts as an internal reference, were co-transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen, USA). Six hours later, newly egressed tachyzoites were harvested, counted and added into the HEK293T cells as indicated; 24-h post parasites infection, cells were collected and lysed, and luciferase activities were measured as recommended (Promega, USA). To test the influence of RHΔHX on NF-κB signaling, HEK293T cells were infected with RHΔHX parasites; ME49 was used as a positive control. Eighteen hours post-infection, human recombinant TNF-α was added at a final concentration of 20 ng/ml and lasted for 6 h. Then, luciferase activities were detected as described above. HEK293T cells were co-transfected with NF-κB and pRL-TK luciferase reporter plasmid along with each of the GRA15-containing plasmids, and after 24 h, the luciferase activity was measured in cell lysates and all repeated three times independently.