Fig 2: Several initiation factors are directly targeted by miR‐322/miR‐503. (A) Putative miR‐322/miR‐503 recognition sites on the 3′‐UTR of eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M, as predicted by TargetScan. (B) miR‐322/miR‐503 inhibited luciferase activity of a firefly luciferase reporter fused with 3′‐UTRs from eIF4G1, eIF4B, eIF2B5, and eIF3M, but not eIF4E. Mutation of the putative recognition sites abolished the inhibition. (C) Confirmation of the down‐regulation of eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M in additional transgenic muscles by RT‐PCR (n = 5 vs. 5). (D) Expression of eIF4E, phospho‐eIF4E, eIF4G1, and eIF4B proteins was down‐regulated in transgenic muscles. The expression of phospho‐eIF2α was unchanged. (E) Quantification of protein expression levels of eIFs (n = 4 vs. 4). (F) New protein synthesis was inhibited in transgenic muscles as determined by SUnSET (surface sensing of translation). Mice were labelled with 0.04 μmol/g puromycin for 30 min. (G) Quantification of levels of puromycin‐tagged peptides (n = 4 vs. 4). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Fig 3: Inactivation of the H19X locus promoted muscle growth and rendered resistance to starvation‐induced atrophy. (A) Diagram of the insertion mutation that blocks downstream transcription in the H19X locus. (B) Quantification of miR‐322, miR‐503, and miR‐542 expression in mutant vs. control muscles (n = 4 vs. 6). (C) Mutant animals had higher body weights than control animals at 6 months old (n = 4 vs. 6). (D) Hindlimb muscle masses were higher in mutant animals than in control animals (n = 4 vs. 6). (E) Frequency histograms of CSA in mutant vs. control muscles (n = 5 vs. 5). (F) Representative images of SDH staining. (G) Percentage of oxidative fibres as determined by SDH staining (n = 11 vs. 11). (H) Quantification of eIF4E, eIF4B, and eIF3M expression in mutant vs. control muscles (n = 4 vs. 6). (I) New protein synthesis was up‐regulated in mutant muscles. (J) Body weight loss was comparable in mutant and control animals after 48 h starvation (n = 9 vs. 11). (K) Interruption of the H19X locus partially rescued starvation‐induced muscle wasting (n = 10 vs. 11). Shown are muscle weight/body weight ratios of starved animals normalized to that of regularly fed control littermates.

Fig 4: Effects of the order of concurrent training on the signaling protein in the gastrocnemius muscle. Representative immunoblots are shown in (a). Phosphorylation and protein expression levels of (b) eIF‐4B, (c) GSK3B, (d) Erk1/2, and (e) p38 in the gastrocnemius muscle after 3 weeks of concurrent training were analyzed by western blotting. Values represent means ± SE

Fig 5: SDOS localizes on the ER and associates with actively translating polyribosomes. (A) Subcellular fractionation of HeLa cells into mitochondrial (Mito), cytosolic (Cyto) and microsomal (ER) fractions following 24 h induction of SDOS-eGFP. Total lysate and fractions were analyzed by WB with indicated antibodies. BiP is used as a ER marker, GAPDH as a cytosolic marker and VDAC1 as a mitochondrial marker. (B) WB analysis of TRAP1-IP from the ER fraction with SDOS, TRAP1 antibodies. Vinculin is used as internal control. (C) Ribosomal purification from HCT116 cells and HCT116 cells transfected with SDOS-myc, followed by immunoblot with ß-ACTIN (as a cytosolic marker), ßF1ATPase (as a mitochondrial marker), RPL3 (as a ribosomal marker) and SDOS antibodies. (D) Polysome profiling absorbance, measured at 254 nm, indicates the sedimentation of the particles: fractions 1 and 2 free cytosolic proteins or light complexes; fractions from 3 to 7 ribosomal subunits (60S, 40S) and monomer (80S); fractions from 8 to 12 polysomes. Proteins from each fraction were analyzed by WB with the indicated antibodies. rpL3 was used as a ribosome marker, eIF4B and eIF4E were used as markers of translation initiation complex. (E) Representative image of PLA showing the interaction of SDOS-eGFP with phosphorylated (active) ribosomal protein S6 (phospho-rpS6) in HeLa cells following 24 h induction of SDOS-eGFP by using GFP and phospho-rpS6 antibodies. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Cycloheximide treatment (200 µg/ml, 1 h) was used to confirm the causal role of active protein synthesis on the binding.