Dual glow luciferase assay kit

We performed luciferase reporter assay on HEK293 cells. These cells were transfected with either GFP-tagged miR-133a (cat # MmiR3445-MR03) or GFP-tagged scrambled miRNA (cat# CmiR0001-MR03), which were purchased from GeneCopoeia, Rockville, MD, USA. We received custom designed CSE 3′ untranslated region (UTR) clones (WT 3′UTR: cat # MmiT038666-MT06; mutant 3′UTR: CS-MmiT038666-MT06-01) from GeneCopoeia, Rockville, MD, USA. For luciferase reporter assay we treated 1 μg of WT or mutant CSE to either GFP miR-133a or GFP-scrambled miRNA expressing cells for 48 hours, and measured the relative luciferase activity using Dual-Glow Luciferase Assay kit (cat # E2920, Promega Corp., Madison, WI) and a GloMax-Multi + Detection System (Promega) following the manufacturer’s manual.

Molecular biology-grade reagents were purchased commercially. Poly-L lysine, protease inhibitor cocktail, H₂DCFDA, DAPI, Flouramaount, SMCC (Succinimidyl- trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate), HIV-TAT1, Iodocetamide, DMF, Cyclodextrin, Cycloheximide (CHX), Etoposide, Methyl β-cyclodextrin (MβCD), anti-His (SAB4301134), trypan blue, and a BCA protein estimation kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni⁺²-NTA beads, LipofectamineLTX plus transfection reagent Prestoblue viability assay kit, IL-1 beta Human ELISA Kit (BMS224HS), and Alexa fluors were purchased from Invitrogen (Life Technologies, Carlsbad, California, USA). A dual glow luciferase assay kit was purchased from Promega (Madison, WI, USA). The death receptor ligands CD 95L and TNF-α were obtained from ProSpec (Rehovot, Israel). HA14-1 was obtained from Maybridge (Cornwall, UK). The details of the antibodies used in this study are provided in Supplementary Material Table S1. All other chemicals used were of analytical grade and purchased from Merck (Darmstadt, Germany).

5 × 10⁴ cells were seeded in 24-well plates. After 24 h, cells were transfected with 500 ng pGLHIF1.3^{47 (link)} and 100 ng pGL4.74 renilla luciferase (#E6921, Promega) per well. After incubation for further 72 h, cells were lysed and prepared using the Dual Glow luciferase assay kit (Promega). Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values to exclude variations in transfection.

We performed a miRNA target validation luciferase reporter assay using H9c2 cells. In brief, cells were transfected with lentiviruses either GFP-tagged miR-18a-5p (cat# RmiR6078) or scrambled miRNA (cat# CmiR0001), which were purchased from GeneCopoeia. We received custom-designed HIF-1α 3′ untranslated region (UTR) clones (WT 3′UTR: cat# RmiT050798; mutant 3′UTR: cat# CS-RmiT050798) from GeneCopoeia, Rockville, MD, United States. For luciferase reporter assay, we co-treated 1 μg of WT or mutant HIF-1α 3′UTR clones with either miR-18a-5p or GFP-scrambled overexpression for 48 h, and measured the relative firefly to Renilla luciferase activity using a Dual-Glow Luciferase Assay kit (cat# E2920, Promega Corp., Madison, WI, United States) and a GloMax-Multi + Detection System (Promega) following the manufacturer’s manual.