Figure 1.
Expression of eIF4AI and eIF4AII during C2C12 differentiation.
(A) Phase contrast images of C2C12 cells grown in the presence of DM for the indicated number of days (d). Scale bars represent 50 µm. (B) Western Blot analysis documenting expression levels of the indicated proteins during C2C12 cell differentiation. Long (l.e.) and short (s.e.) exposures of the eIF4AII Western blot are presented. (C) Quantification of changes in eIF4AI and eIF4AII protein levels relative to those obtained on day 0. n = 3±SEM. (D) 35S-methionine/cysteine incorporation into TCA-insoluble protein. C2C12 cells were induced to differentiate and protein extracts were prepared at the indicated time points. Cells were labeled for 30 min and the amount of radiolabeled protein quantitated by TCA precipitation. Values are standardized against total protein content. n = 3±SEM. (E) eIF4AI/II are efficiently incorporated into the eIF4F complex during C2C12 differentiation. m7GTP affinity purification of the eIF4F complex from C2C12 cells at the indicated days following induction of differentiation. Western blots to the indicated proteins were performed on an aliquot of input extract (lanes 1–4), GDP eluents (lanes 5–8), and m7GTP eluents (lanes 9–12).
Figure 2.
Expression of eIF4AI and eIF4AII during primary myoblast differentiation.
(A) Phase contrast images of primary myoblasts induced for differentiation over the indicated number of days (d). Scale bars represent 50 µm. (B) Western Blot analysis documenting expression levels of the indicated proteins during primary myoblast differentiation. (C) Quantification of changes in eIF4AI and eIF4AII protein levels relative to β-actin. n = 3±SEM.
Figure 3.
Transcriptional changes in eIF4AII mRNA levels during C2C12 differentiation.
(A) Changes in eIF4AI and eIF4AII mRNA levels during C2C12 cell differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 3±SEM. (B) Transcriptional changes in eIF4AI and eIF4AII mRNA levels during primary myoblast differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 4±SEM. (C) Nuclear Run-On analysis of GAPDH, MyoD and eIF4AII transcription in C2C12 cells at days 0 and 3 after induction of differentiation. Probes targeting the 5′ and 3′ UTRs of eIF4AII were used to distinguish the transcript from that of eIF4AI. (D) Quantiation of nuclear run-on experiments. Changes in eIF4AII transcription was quantified using a Typhoon Scanner (GE Healthcare) (values are normalized to GAPDH mRNA levels which did not change over this time period).
Figure 4.
MyoD binds to the endogenous eIF4AII promoter following induction of C2C12 differentiation.
(A) Schematic representation of the eIF4A and eIF4AII promoters showing the relative position and nucleotide sequence of putative MyoD binding sites. Positions are relative to the transcription start site (+1). Arrows denote the relative position of the primers used in the ChIP assay. (B) ChIP assays performed with C2C12 extracts prepared on the indicated days following induction of differentiation. Equivalent amounts of crosslinked chromatin were immunoprecipitated using either an anti-MyoD antibody or an IgG control. The presence of eIF4AI and eIF4AII promoter sequences in the immunoprecipitations was evaluated by qPCR. Primers to the myogenin promoter were used as control and values are normalized to input levels. The input sample represents 5% of the initial DNA material following sonication before immunoprecipitation. n = 4±SEM. (C) Products of qPCRs from ChIP assays performed in (B) (In-Input, Ig- IgG elution, MD- MyoD elution).
Figure 5.
The proximal eIF4AII promoter is activated by MyoD.
(A) Schematic representation of Renilla Luciferase reporters linked to eIF4AI and eIF4AII proximal promoter sequences. The relative position of putative MyoD binding sites is indicated. (B) Transactivation assays involving eIF4AI and eIF4AII reporter constructs and MyoD expression vectors. NIH-3T3 cells were transfected as indicated in the Materials and Methods and extracts prepared on the indicated days following differentiation. Relative light units (RLU) were standardized to protein levels. The ratio of MyoD dependent expression relative to empty expression vector is plotted. n = 4±SEM.
Figure 6.
C2C12 differentiation is curtailed upon suppression of eIF4AI and eIF4AII.
(A) Western blot analysis of siRNA- or hippuristanol- treated C2C12 cells at the indicated times following induction of differentiation. Blots were probed with antibodies to the proteins indicated to the left of the panels. (B) Phase contrast images of siRNA- or hippuristanol- (125 nM) treated C2C12 myoblasts at initiation (Day 0) or three days post-induction of differentiation. Scale bars represent 50 µm. (C) Immunofluorescence images of siRNAs or hippuristanol treated C2C12 cells at day 3 after induction of differentiation. Myofiber formation was analyzed by immunofluorescence using anti-myosin HC and anti-myoglobin antibodies. Scale bars represent 50 µm. (D) Fusion index of muscle fibers (% Differentiation). n = 3±SEM. (E) Quantification of myofibers/field. n = 3±SEM.