Cignal reporter assay

SSCT fibroblasts were stably transfected with a reporter plasmid containing the Smad binding element. The Smad binding element comprises a CAGACA motif that serves as a direct binding site for Smad proteins using the Cignal Reporter Assay (Qiagen, Valencia, CA, CCS-017L) as the Smad binding element. Cells were plated at 1.3×10⁵ fibroblast cells per well and the following day cells were transfected with pathway reporters using FuGENE 6 transfection reagent (Promega Corporation, Madison, WI, E2691) according to the manufacturer’s protocols. After 24 hours culture, the transfected cells were pretreated with each inhibitor for 60 minutes and then treated with ± TGF-β (5ng/mL) as noted. After 24 hours cells were lysed with passive lysis buffer (Promega Corporation, Madison, WI, E1941) and cell lysates luciferase activity was measured upon addition of luciferase assay reagent (Promega Corporation, Madison, WI, E1483) using the Dual-Luciferase® Reporter Assay System (Promega Corporation, Madison, WI, E1910) as previously reported (Gingery et al., 2014 (link)). Relative units of luciferase activity were reported after subtracting the basal expression levels observed in the Veh group.

SSCT fibroblasts were transfected with a reporter plasmid containing the Smad binding element. The Smad binding element comprises a CAGACA motif that serves as a direct binding site for Smad proteins using the Cignal Reporter Assay (Qiagen, Valencia, CA, USA) as the Smad binding element. Cells were plated on 48-well plates (1.3×10⁵ per well) and the following day cells were transfected with pathway reporters using FuGENE 6 transfection reagent (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocols. After 24 hours culture, the transfected cells were pretreated with Bindarit (300 μM) and/or SD208 (1 μM) for 60 minutes and then treated with ± TGF-β1 (5 ng/mL) as per experimental protocol. The concentration of SD208 was determined according to the previous report [9 (link)]. After 24 hours cells were lysed with passive lysis buffer (Promega Corporation, Madison, WI, USA) and cell lysates luciferase activity was measured upon addition of luciferase assay reagent (Promega Corporation, Madison, WI, USA) using the Dual-Luciferase® Reporter Assay System (Promega Corporation, Madison, WI, USA) as previously reported [7 (link)]. Relative units of luciferase activity were reported after subtracting the basal expression levels observed in the Veh group.

293T Lenti-X cells were seeded in triplicate at 2.25×10⁵ cells/well in 24 well plates. For the NF-kB reporter assay, cells transfected with either pCDH-MCS1-mCherry-Rgnef, or pCDH-MCS1-GFP-p190RhoGEF Y1003A and either pMIL-6 FL or pmIL-6 mut NF-kB using Fugene HD (Promega) or jetPRIME (Polyplus). For inhibitor treatments, 30 min before transfection, cells were treated with vehicle or 10 μM Bay 11–7082, or 10 μM TPCA-1 and further incubated for 24 h. The ARE (antioxidant response element) Cignal™ Reporter Assay (Qiagen) was performed according to standard manufacturer instructions. Dual-Luciferase Reporter Assay system (Promega) was carried out 24h after transfection according to the manufacturer’s protocol using a Synergy HTX multi-mode plate reader (BioTek). As an internal control of transfection efficiency, the renilla luciferase encoding plasmid (pRL-TK, Promega) was co-transfected and for each sample, firefly luciferase activity was normalized to the renilla luciferase activity.

We generated K562, NALM-6 SF3B1 isogenic cells,
HAP1 and CASP8^KO HAP1 cells expressing the luciferase reporter
for NF-κB response elements by following the manufacturer
instructions (Cignal™ Reporter Assay, Qiagen). Cells were stimulated
with LPS, TNFα or TRAIL as described above, and NF-κB activity
was assessed by luciferase intensity using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer instructions. To verify
that the luciferase reporter assay was not aberrantly activated by basal
leakiness, we used another NF-κB reporter plasmids (Promega; N1111)
with known NF-κB response elements (RE), and performed mutagenesis in
the NF-κB-RE using the QuiK Change II Site-Directed Mutagenesis Kit
(Agilent Technologies; #200522). There are five putative NF-κB-RE
binding sites in this reporter plasmid (5′-GGGRNTTTCC-3′,
where R is a purine, Y is a pyrimidine and N is any nucleotide). To
mutagenize the binding sequence, the “TTTC” sequence was
mutated to “AAAA”. The primers used to create the two mutant
plasmids are: