Fig 1: eIF4E regulates the expression of HA synthesizing enzymes and HA receptor CD44.(A) HA biosynthesis pathway. (B) RT-qPCR of HA synthesizing enzymes and its receptor CD44 following RNA immunoprecipitation (RIP) using anti-eIF4E antibody from nuclei of U2Os 2Flag-eIF4E cells. Data are normalized to IgG control and presented as fold change. c-Myc and Mcl-1 are positive targets of eIF4E and thus serve as positive controls, whereas GAPDH and 18S rRNA served as negative controls. We used standard deviation to denote statistical significance. One representative experiment is shown, which was carried out in triplicate. (C) RT-qPCR of HA synthesizing enzymes in cytoplasmic versus nuclear fractions of U2Os cells overexpressing 2Flag-eIF4E (2F4E), S53A mutant (2FS53A) or vector control (2Fvect). Data are normalized to vector control. c-Myc and Mcl-1 served as known eIF4E targets, whereas GAPDH, Hsp90 and ß-Tubulin served as negative controls. One representative experiment is shown, which was carried out in triplicate. (D) Western blot of HA synthesizing enzymes and CD44 as a function of eIF4E or S53A mutant overexpression. Mcl-1 served as positive eIF4E target control. Actin was used as a loading control. Each Actin blot corresponds to the western blots above it. Both 2Flag-eIF4E and endogenous eIF4E are shown. HK: Hexokinase; HAS3: Hyaluronan Synthase 3; PGM5: Phosphoglucomutase 5; UGP2: UDP glucose pyrophosphorylase; UGDH: UDP glucose dehydrogenase; GFPT1: Glutamine fructose 6 phospho transaminase; GNPNAT1: Glucosamine phosphate N-acetyltransferase; UAP1: UDP N-acetyl pyrophosphorylase; GPI: Glucose-6-phosphate isomerase; CD44: HA receptor; MMP9: Matrix Metalloproteinase 9. For bar graphs, the mean ± standard deviation are shown. Experiments were carried out in triplicate, at least three independent times.