Figure 6.
Changes in the ratio of STAU1 and PKR during hSkMc cell myogenesis alters LIN28 mRNA translation via its 3′ UTR IRAlus, which is key to myogenesis. (A) Western blotting of hSkMc cell lysates. Cells (2 × 106 per 150-mm dish) were propagated in growth medium to 80% confluency. An aliquot was harvested (0 d in differentiation medium [DM]), and the remaining cells were propagated in DM and harvested on the specified day. (Myosin HC) Myosin heavy chain. Ponceau S-staining demonstrated that equal amounts of lysates were analyzed. (B) Histogram representation of RT-qPCR quantitations (Supplemental Fig. S6G) of RNA from samples analyzed in A. The level of each mRNA was normalized to the level of U6 snRNA, and the normalized level in MBs (day 0) is defined as 100. (C) Line graph representation of the denoted ratios using samples analyzed in A and B. Each ratio in MBs (day 0) is defined as 100. (D) Western blotting of lysates of hSkMc MBs (2 × 106 per 150-mm dish), which had been transiently transfected with 1 μg of pcDNA3-Flag-LIN28Δ3′UTR and, 3 d later, cultured for the specified number of days in DM. (E) Differential interference contrast microscopy of hSkMc cells analyzed in A and D. All results are representative of three independently performed experiments.