Compound 3

For HepG2 cell immunofluorescence (IF) microscopy, cells were plated on poly L-lysine coated glass coverslips and fixed after treatment with dimethyloxaloylglycine (500 μM) or Compound 3 (50 μM) (Sigma-Aldrich, St. Louis, MO, USA) using 4% paraformaldehyde. Cells were permeabilized using 0.25% TRITON X-100 in phosphate-buffered saline (PBS) × 10 minutes and blocked with Background Sniper (Biocare Medical, Pacheco, CA, USA). Primary antibodies diluted in Dako Diluent against phosphorylated IGFBP-1 at Ser101 (1:400), Ser119 (1:400), or Ser169 (1:200), polyclonal IGFBP-1 (1:2500, a kind gift from Dr. R. Baxter, Sydney, Australia), or PKCα (1:250) and incubated overnight at 4°C. The Alexa Fluor fluorescent secondary antibodies used were diluted in Dako Diluent (1:400) anti-mouse 488 or anti-rabbit 568 (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 45 minutes at room temperature. Samples were counterstained with DAPI (1:300). The stained coverslips were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA).

HepG2 cells grown to 75% confluency were serum-starved in 2% FBS overnight prior to treatment of cells with echinomycin (Sigma-Aldrich, St. Louis, MO, USA), dimethyloxaloylglycine (DMOG, Cayman Chemical Company, Ann Arbor, MI, USA) or Compound 3 (Sigma-Aldrich, St. Louis, MO, USA). echinomycin is an inhibitor of HIF-1 activity, by blocking the binding of HIF-1 to DNA and preventing its activity as a transcription factor. DMOG increases the expression of HIF proteins by inhibiting their breakdown. Compound 3 (C3) is an inducer of REDD-1. Dose and time dependency studies were performed to identify the optimal drug concentration (echinomycin 10 nM, DMOG 500 μM, and C3 50 μM) and time of incubation (24 hours) used in this study to maximize effects while minimizing cell death (not shown). Prior to treatment, media was changed to 2% FBS overnight (12–16 hours). Cells were subjected to inhibitor/activator treatments in normoxic or hypoxic conditions for 24 hours. Subsequently, cell media was collected and immediately stored at −80°C and cells were stored at −80°C prior to collection of lysate as described previously^{28 (link)}.

MC was generously gifted by Professor Maria Tomasz. Reagents for the chemical synthesis and purification of DMC, mitomycin A and compound 3 were purchased from Sigma Life Sciences (St. Louis, MO) unless otherwise stated. Phosphodiesterase I (snake venom diesterase (SVD), Crotalus adamanteus venom, E.C. 3.1.4.1.) and alkaline phosphatase (Escherichia coli, EC 3.1.3.1) were obtained from Worthington Biochemical Corp (Freehold, NJ). Nuclease P₁ (penicillium citrinum, EC 3.1.30.1) was obtained from Sigma Life Sciences (St. Louis, MO). Sep-Pak C-18 cartridges were purchased from Waters Corp (Milford, MA). Oligonucleotides were purchased from Midland Certified Reagent (Midland, TX). M. lysodeicticus DNA (M. luteus DNA, type XI; 72% GC) and calf thymus DNA (type XV; 42% GC) were from Sigma Life Sciences (St. Louis, MO).