Dual glo

MEISi-1 (MW: 388.467) (Cas# 446306-43-0) and MEISi-2 (MW: 306.32) (Cas#2250156-71-7) are now available by Meinox Pharma Technologies (www.meinoxtech.com, Cat# MEISi1-25mg and Cat# MEISi2-25mg). Lyophilized MEISi-1 and MEISi-2 small molecules were dissolved in sterile dimethyl sulfoxide (DMSO, Calbiochem, 317275). Luciferase reporter assays were carried out as we have done previously^{13 (link),14 (link),16 (link),17 (link)}. Briefly, p21-pGL2 (2 µg) (MEIS-p21-Luc reporter^{17 (link)}) or Hif-pGL2 (MEIS-HIF-Luc Reporter or PBX-Luc reporter (ref.^{13 (link),14 (link),16 (link)}) was co-transfected with Meis1 expression vector pCMVSPORT6-Meis1 or Pbx1 pCMVSPORT6-Pbx1 (OpenBiosystems, 400 ng), pGL2 (filler) into HEK293 cells in six-well plates using poliethylenimine transfection reagent (SantaCruz, Cat.No. Sc-360988A). DMSO (control, 0.5%), MEISi-1 and 2 were tested in three different doses (0.1, 1 and 10 µM) in triplicates. After 48 hours of transfection, the cell lysate was processed for luciferase activity using the luciferase reporter system, according to the manufacturer’s instructions (Promega Dual-Glo, Cat.No. E2920). Luciferase measurements were done with Luminoskan Ascent Microplate (Thermo Lab System) and were calculated as relative light units normalized to transfection control/protein content.

Yeast cells transformed with a plasmid encoding for RNA2-Rluc reporter (pJJ-16) were grown as described in Yeast Cultures section. When they reached an OD₆₀₀ of 0.5, protein synthesis was stopped with 0.5 mg/ml cycloheximide. Renilla luciferase activity assay (Dual-Glo®, Promega) was used following the protocol provided by the manufacturer. Samples were collected at different intervals during 3 h by directly transferring 10 µl of culture to 100 µl of 1x Passive Lysis Buffer. Only 10 µl of the lysate were used (the rest was stored at −80 °C) and 200 µl of LARII-StopGlo solution (1:1) was subsequently added. FB12 Luminometer was employed to read Luciferase activity, with 5 s of equilibration time and 5 s of measurement time. The values obtained were corrected by the corresponding OD₆₀₀ and were represented relatively to the first time-point (t = 0). This protocol was adapted from ref. ^{46 (link)}.

The mRNA reporters used in cell reporter assay contained the 5’-untranslated region (5’-UTR) of β-globin (HBB) followed by either the stalling sequence of PCSK9 (codons for amino acids 1–35), the p2A sequence [50 (link)] and Renilla luciferase (PCSK9(1–35)-Rluc), or by just the Firefly luciferase sequence (Fluc). Briefly, 125 μL of K562 cells were seeded in 96-well plates at ~0.4x10⁶ cells/mL and incubated overnight (37°C, 5% CO₂). The test reporter (PCSK9(1–35)-Rluc) and the control reporter (Fluc) were co-transfected (0.05 μg per mRNA per well) in K562 cell lines using JetMessenger mRNA transfection kit (Polyplus, 150–01) according to the manufacturer's instructions. Serial dilutions of 200x PF8503 were prepared in DMSO and added to the cells immediately after transfection (0.5% DMSO final per well). Rluc and Fluc luminescence signals were measured 7–8 hr after transfection with Dual-Glo (Promega, E2920) on a Veritas microplate luminometer (Turner Biosystems). The normalized signal for each well (Rluc/Fluc) was calculated and normalized to the signal of non treated (DMSO only) wells. IC50 values were calculated using GraphPad Prism version 7.00 for Mac (GraphPad Software, La Jolla California USA, www.graphpad.com).

McArdle cells were transfected with a pGl3 reporter vector (Promega) containing the mouse Fndc4 upstream promoter region using Lipofectamine 2000 (Life Technologies) as transfection reagent. After 8 h, cells were treated with TGF-β1 (20 ng ml⁻¹; Peprotech, Stockholm, Sweden) and/or SB-431542 (20 μM; Merck Millipore) for 20 h. The luciferase assay (Dual-Glo, Promega) was performed according to the manufactures instructions. Values are reported relative to the non-treated control group.

The SMN2 reporter cell line [14 (link), 15 (link)] was cultured in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1X Penicillin/Streptomycin. Reporter cells media was supplemented with hygromycin during maintenance. All cells were maintained at 37°C and 5°C CO₂. After seeding in 96-well white tissue culture plates at 25,000 per well, cells were incubated overnight before being exposed to different doses of compounds or DMSO for 24 hrs. The final DMSO concentration was kept constant at 0.1% (v/v). Firefly and renilla luciferase expression were assayed with DualGlo (Promega E2920) and measured on a PHERAstar FS microplate reader (BMG Labtech). Relative light units are normalized to DMSO control and expressed as a percentage.

For the luciferase reporter assay, the cells were co-transfected with the NF-κB promoter-driven luciferase reporter (0.2 μg) and 0.01 μg of Renilla luciferase-expressing plasmid (pRL-TK; Promega, Madison, WI, USA) using the GeneJammer transfection reagent (Stratagene, La Jolla, CA, USA). Twenty-four h after the transfection, the cells were treated with LPS for 24 h with or without a 1-h pretreatment of kallistatin (0.5 μM) and NAC (10 mM). Cells were then lysed and harvested for luciferase and Renilla measurement using a luciferase assay system (Dual-Glo; Promega). For each lysate, the firefly luciferase activity was normalized to the Renilla luciferase activity to assess transfection efficiencies.