Renilla construct

Vectors with the luciferase gene and the native 3’untranslated region (3’UTR) construct of TRAF6 or IRAK1, described in [34 (link)], were purchased from AddGene (www.Addgene.org). The native iNOS promoter construct was described in [35 (link)], and the NF-κB promoter construct (pNF-κB) containing a repetitive κB motive was purchased from Stratagene. The miR-146a-5p promoter construct, described in [34 (link)], was also ordered from AddGene. The luciferase activity was measured by a dual luciferase reporter assay system (Promega), according to the manufacturer’s protocol. The luciferase activity was normalized to a co-transfected vector (pRL-TK) containing a HSV-TK promoter driven Renilla construct (Promega). Readings were conducted on a luminometer from Berthold Technologies.

Embryos were electroporated with the DNAs indicated together with a NeuroDp-Luciferase reporter (Saade et al., 2013 (link)) and a renilla-construct (Promega) for normalization. GFP-positive neural tubes were dissected out at 48 hr after electroporation and homogenized in passive lysis buffer. Firefly- and renilla-luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega), and the data are represented as the mean ±sem from at least 14 embryos per experimental condition.

Cells (50,000/well) were plated in 24-well plates and transfected with 1 μg/well of reporter constructs containing PUMA (26 (link)) (4XBS2 WT Catalog # 16593, Addgene), p21 (PG13-Luc, Catalog # 16442, Addgene), or GADD45A (GADD45 WT-Luc, Catalog # 8356, Addgene); promoters harboring p53-binding sites were transfected using Lipofectamine 2000 (Invitrogen). Renilla construct (25 ng/well, Promega) was cotransfected as a control for transfection efficiency. Approximately 24 h post transfection, A549 cells were treated with 2.0 μM NMDi for 17 h. Luciferase assay was performed using Dual-Luciferase Assay System (Catalog # E1960, Promega) according to manufacturer’s instructions. Luminescence was measured using GLOMAX 20/20 Luminometer (Promega). Luciferase activity was normalized with Renilla activity.
For assessing promoter-binding ability of p53-depleted A549 cells, cells transfected with either siControl or sip53 (50,000 cells/well) were used and the experiment was performed as described above.

SV40 large T-antigen-expressing HEK cells (293T cells) were plated onto 24-well
plates. Next day, they were transfected with 2 μg Lef1 reporter
construct using Lipofectamine 2000 reagent and 0.02 μg Renilla
luciferase construct (Promega, Madison, WI, USA) for 6 h in the absence of
serum.^{40 (link)} In some cases, the cells
were co-transfected with siRNA oligonucleotides targeting the test genes
PDE5a, PKG1, PKG2, and GSK3β (see
Supplementary Table 1 for the specific sequence
information), Lef1 reporter construct and Renilla construct. Transfectants were
then cultured in a 1 : 2 diluted conditioned medium prepared from
control L cells or Wnt3a-expressing cells in the presence of tadalafil and/or
vardenafil for 48 h. At the end of the designated culture, cell lysates
were prepared and dual-luciferase assay was performed according to the
manufacturer's instructions (Promega). The firefly luciferase activity was
normalized to the Renilla luciferase activity.

Embryos were electroporated at HH stage 11–12 with the indicated DNA, together with a TOPFLASH luciferase reporter construct containing synthetic TCF-binding sites (Korinek et al., 1998 (link)) as well as a Renilla construct (Promega) for normalization. Embryos were harvested after 24 hpe and GFP-positive neural tubes were dissected and homogenized with a douncer in passive lysis buffer (Promega). Firefly- and Renilla-luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega). Data were obtained from at least two independent experiments (n = 6–12 embryos per experimental condition).