Figure 3. eIF4E elevates HA in cancer cell lines and primary specimens.

(A) RT-qPCR of HA synthesizing enzymes and its receptor CD44 following RNA immunoprecipitation (RIP) from nuclei of MM-6 cells using anti-eIF4E antibody. Data are presented as fold change relative to IgG controls. c-Myc and Mcl-1 served as endogenous eIF4E targets, while GAPDH, β-Tubulin and 18SrRNA served as a negative control. (B) RT-qPCR of HA synthesizing enzymes in cytoplasmic versus nuclear fractions of MM-6 cells treated with Ribavirin (Rib). Data are normalized to untreated control. Error bars are means ± S.D. c-Myc and Mcl-1 served as positive controls since they are known eIF4E targets, while GAPDH, β-Tubulin, RPIIa and Survivin served as negative controls. (C) Western blot of HA synthesizing enzymes and CD44 as a function of Ribavirin treatment in MM-6 cell line. Mcl-1 served as endogenous eIF4E target, while Hsp90 served as a loading control. (D) Fluorescence staining of HA (in red) in MM-6 cell lines treated with Ribavirin (Rib) versus untreated (UN) in the presence or absence of Hyaluronidase treatment. DAPI is in blue. A 63x objective with no digital zoom was used. (E) Fluorescence staining of HA (in red) in human CD34+ specimens from healthy volunteer compared with leukemic cells from M5 AML Patient. (F) Quantification of HA fluorescence staining in 9 M4/M5 AML patients and CD34+ specimens from five healthy volunteers using ZEN software. HA signal intensity is presented as the geometric means of the HA signal. The mean ± standard deviations are shown. (G) Fluorescence staining of HA (in green) in 66cl4 cells in the presence or absence of Hyaluronidase or Ribavirin treatment. DAPI is in blue. A × 63 objective with no digital zoom used. (H) Western blot control of HAS3 and CD44 as a function of ribavirin treatment in 66cl4 cell line. Mcl-1 served as endogenous eIF4E target, while ActinB served as loading control. Experiments were carried out in triplicate, at least three independent times. For bar graphs, the mean ±standard deviation are shown.

Figure 3—figure supplement 1. eIF4E elevates HA in cancer cell lines and primary specimens.

(A) Control western blot for cellular fractionation corresponding to eIF4E RIP. Lamin and Pol II are used as nuclear markers while Raf1 is used as cytoplasmic marker. (B) Control western blot of eIF4E recovered in immune complexes with eIF4E antibodies and IgG relative to input amount of eIF4E corresponding for RIP in MM6 cells in Figure 3. (C) RT-qPCR of total mRNA levels corresponding to mRNA export assay in untreated cells (UN) or Ribavirin treated (Rib). (D) Semi-qPCR for tRNAlys and U6 snRNA served as controls for the cytoplasmic and nuclear fractions, respectively, for Figure 3.

Figure 3—figure supplement 2. eIF4E concentrates in HA rich protrusions and correlates with sites of active translation.

(A–B) Fluorescence staining of Click_iT HPG Alexa Flour 488 in 66cl4 or U2Os cells overexpressing eIF4E cells (2F4E), respectively, following treatment with 20 µM Ribavirin versus untreated control. Cyclohexamide treatment (+CHX) is used as a negative control. A x40 or 63 objectives is used. Experiments were carried out in triplicate, at least three independent times.