Dual luminescence assay kit

The TNNT2 (HPRM12846-PG04) and NPPA (natriuretic peptide A; HPRM23486-PG04) promoter activities were measured using a Dual Luminescence Assay Kit with a dual reporter construct containing Gaussia luciferase (GLuc) and secreted alkaline phosphatase (SEAP) side by side from a single sample (all from GeneCopoeia, Inc.). Each promoter is placed upstream of the GLuc reporter gene and contains a specific cardiac transcription factor as an insert. A secondary reporter gene SEAP was used to monitor the transfection efficiency for normalization. To detect serum response element (SRE) promoter activities, both SRE fragment (pGL4.33 [luc2P/SRE/Hygro], Promega) and pGL4.74 (hRluc/TK, Promega) were co-transfected into human cells by using X-tremeGene HP reagent (Roche). The relative expression of SRE (luc2P) construct was normalized using a control vector (hRluc) introduced into the same cells. Human cells were transfected with human NKX2-5 cDNA (SC122678), human NKX2-5 shRNA (short hairpin RNA; TR311165B), human HAND1 cDNA (SC122690), human HAND1 shRNA (TR316857C), human NOTCH1 cDNA (SC308883), or human NOTCH1 shRNA (TR302916D) along with single- or dual-reporter constructs (all from OriGene Technologies, Inc.). The luciferase activities were measured by the Glomax-Multi+Detection System (Promega) 48 hours after transfection.

For the dual luciferase assay, LNCaP and C4-2 cells were plated into 12-well plates and co-transfected with 1 μg of the seap reporter gene construct and 15 pmol of YAP1 luciferase together with siRNA of other plasmids by using transfection reagent (Roche). Transfected cells were cultured and 24 h later the supernatants were collected for luciferase assay using Dual Luminescence assay kit (GeneCopoeia MD) according to the manufacturer’s instructions. To measure SOX2 and Nanog promoter driven luciferase activity, the Glo Luciferase Assay System (Promega) was used.

Apigenin was purchased from Aladdin (Los Angeles, USA). Crystal violet and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sangon Biotech (Shanghai, China). Matrigel and transwell chambers were purchased from BD Biosciences (San Jose, CA, USA). The antibodies for claudin, N-cadherin, vimentin, and E-cadherin were purchased from Affinity Bioreagents (Colorado, USA). An immunofluorescence staining kit with FITC- and TRITC-labeled goat anti-rabbit IgG and cytoskeleton red fluorescent probe ActinRed were purchased from KeyGEN BioTECH (Nanjing, China). Fluorescein isothiocyanate AffiniPure Goat Anti-Rabbit IgG (H + L) secondary antibodies were purchased from EarthOx (San Francisco, CA, USA). A dual luminescence assay kit was purchased from GeneCopoeia (Guangzhou, China).

PTL was purchased from Aladdin (Shanghai, China). Crystal violet and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sangon Biotech (Shanghai, China). The antibodies to Occludin, Claudin3, EMA, N-cadherin, VEGFR1, CD34, and Vimentin were purchased from Affinity Bioreagents(Colorado, USA). The immunofluorescence staining kit with FITC-labeled goat anti-rabbit IgG and cytoskeleton red fluorescent probe Actin Red were purchased from KeyGENBioTECH (Nanjing, China). A dual luminescence assay kit was purchased from GeneCopoeia (Guangzhou, China).

Snail‐Gluc, Vimentin‐Gluc, E‐cadherin‐Gluc, and GAPDH‐Gluc plasmids were purchased from GeneCopoeia (Guangzhou, Guangdong, China). All plasmids contained SEAP signals. Cells were transfected with the indicated plasmids, and the Dual Luminescence Assay Kit (GeneCopoeia) was used to capture the signals in each well according to the manufacturer's instructions. The ratio of Gluc to SEAP was plotted to graph triplicate results.