Nano glo dual luciferase assay system

To investigate the effect of TMZ on the activity of miR-128 promoters, several reporter constructs were produced. A 2.5-kb fragment of miR-128-1 and miR-128-2 promoters was respectively isolated through PCR by using primers listed in S1 Table. After digestion of the PCR product with MluI and HindIII, the insert was cloned into the pGL3 reporter vector (Promega), creating the expression vectors pGL3-miR-128-1-prom and pGL3-128-2-prom. After sequencing, pGL3-miR-128-1-prom was used as a template, and pGL3-miR-128-2K, pGL3-miR-128-1.5K, pGL3-miR-128-1K, and pGL3-miR-128-0.5K were respectively isolated through PCR. Site-directed mutagenesis of c-Jun-binding sites in miR-128 promoters was performed using the QuikChange® site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) and named pGL3-miR-128-1-prom-MUT, for which pGL3-miR-128-1-prom was used as a template. For reporter assays, cells were transiently transfected with different sizes of wild-type or mutant reporter plasmids and then treated with TMZ using Lipofectamine 3000 (Invitrogen). pNL1.1-TK plasmids were cotransfected and served as an internal control. The reporter assay was performed 24 h after transfection by using the Nano-Glo dual luciferase assay system (Promega).

To confirm that methylation of non-promoter sequences did not significantly affect reporter activity, we obtained the pCpGL plasmid as a kind gift of Dr. Shaohui Hu from the Heng Zu Lab at JHMI. Since the pCpGL reporter plasmid lacks CpG dinucleotides, only promoter sequences will contain CpG residues. The TERT Del 5, TERT Del 5 with C228T, and GSTP1 promoters were cloned into pCpGL to generate 3 unique constructs. The reporter plasmids were treated with SssI or mock, and transfected into cells cultured in 96-well plates. Each well was treated with a transfection mixture containing 11.6 μl Opti-MEM, 54 ng pCpGL vector, 4 ng Renilla (pRL-CMV, Promega), and 0.12 μl X-tremeGENE HP. After 2 days, transfected cells were analyzed for reporter activity using the Nano-Glo Dual Luciferase Assay System (Promega) as described above.

HAP1 or HEK293T cells were transfected with a pcDNA3.1 plasmid encoding NanoLuc or NanoLuc containing artificial intron variants (see Table 1) and pGL4.53[luc2/PGK] vector (Promega) for normalization. Cells were harvested 24–48 h post transfection and analysed using the Nano-Glo Dual Luciferase Assay System (Promega) according to manufacturer’s instructions.

The luminescence of Luc2 and Nluc was measured 24 h after transfection using a Nano-Glo Dual-Luciferase Assay System (Promega K.K., Tokyo, Japan) and a GloMax Navigator microplate luminometer (Promega K.K.).

Sub-confluent SAECs in 48-well plate were co-transfected by UPR luciferase reporter and FLAG-XBP1s expression vector using Lipofectamine 3000 (Invitrogen) according to manufacturer's instruction. Specifically, 100 ng of specific UPR luciferase reporter, 10 ng of the minimal promoter-driven NanoLuc luciferase reporter, pNL3.1 (Promega) as the internal control, and increasing amounts (0 to 300 ng) of FLAG-XBP1s expression vector were used. The difference in total DNA amount was compensated by empty cDNA vector. The UPR luciferase reporters used include pGL4-UPRE-luc2P-Hygro, pGL4-ERSE1-luc2P-Hygro and pGL4-ERSE2-luc2P-Hygro, minimal promoters driven by three copies of previously identified UPR cis-elements, unfolded protein response element (UPRE) and ER stress response element (ERSE)-I and -II (Addgene plasmid numbers 101788, 101789 and 101790, respectively) (18 (link)). The transfected cells were cultured for 48 h prior to harvest in 100 μl of Passive Lysis Buffer (Promega, Fitchberg, WI, USA). Dual luciferase assay was carried out using Nano-Glo Dual Luciferase Assay System (Promega) per manufacturer's instruction. 40 μl of cell lysate was used in each reaction. The firefly luciferase activities produced by the UPR luciferase reporters were normalized by corresponding NanoLuc luciferase activities produced by pNL3.1 and presented as mean ± 25-75% range.