Fig 2: PR20 neither influences the activation of TORC or AMPK nor ER stress. Mixed spinal cord cultures were exposed to PR20 for 48 h and then processed for immunoblotting. A, Representative immunoblot images of biochemical markers of TORC activation (e.g., phospho-4EBP1 and phospho-S6K), AMPK activation, or activation of the ER stress response (KDEL) shows no difference between PR20 versus vehicle-treated cells. B, Quantification of the bands with intensity values from samples were averaged and normalized to actin loading controls. Dark grey bars correspond to the presence of PR20, and light grey bars correspond to levels without PR20. Error bars represent SE.

Fig 3: Overview of the role of mTORC1 signalling in promoting ribosome biogenesis following a single session of resistance exercise, and the potential effect of incorporating endurance training (i.e., performing concurrent training). Adapted with permission from24. Ribosome biogenesis involves transcription of the 45S rRNA (ribosomal RNA) precursor (45S pre-rRNA) (A) mediated by RNA Polymerase I (Pol-I), processing of the 45S pre-rRNA into several smaller rRNAs (18S, 5.8S and 28S rRNAs) (B), assembly of these rRNAs and other ribosomal proteins into ribosomal subunits (40S and 60S) (C), and nuclear export of these ribosomal subunits into the cytoplasm24,26 (D). As well as regulating translational efficiency via downstream control of p70S6K (p70 kDa ribosomal protein subunit kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E binding protein 1) (E), mTORC1 is a key mediator of ribosome biogenesis by regulating transcription factors for genes encoding RNA Pol-I (and also RNA Pol-II and –III, which are not shown in figure)25. Transcription of the 45S pre-rRNA by RNA Pol-I requires a transcriptional complex including TIF-1A (transcription initiation factor 1A; also known as RRN5) and UBF (upstream binding factor), both of which are regulated by the mTORC1 pathway28,29 (F). Activation of AMPK is known to inhibit mTORC1 signalling in rodent skeletal muscle64, and AMPK activation in skeletal muscle is traditionally associated with endurance-type exercise. However, whether signalling events initiated by endurance training, when performed concurrently with resistance training, have the potential to interfere with mTORC1-mediated regulation of ribosome biogenesis is currently unclear (G).

Fig 4: Analysis of AKT/mTORC1 pathway. (A) Western blot analysis of phosphorylated and non-phosphorylated AKT, mTOR, 4EBP1, p70S6k in control and T. cruzi infected cardiomyocytes at 24 hpi. GAPDH was used as loading control. (B) Densitometry analysis of western blots. Values represents the mean ± standard deviation of three or two independent biological replicates (*p < 0.05).

Fig 5: Protein synthesis decreases after DSBs. (A) Polysome profiles of HEK cells treated with 5 µm etoposide or 5 µm etoposide and 200 nm ISRIB for 16 h. (B) Polysome profiles of HEK cells 72 h after electroporation with Cas9-sgIntron or Cas9 without guide (apo Cas9). (C) l-azidohomoalanine (AHA) bulk translation assay demonstrates that DSBs reduce protein synthesis. HEK cells were lysed after 16 h after treatment with 5 µm etoposide and 200 nm ISRIB. Two hours before lysis, growth media was replaced with methionine-free media containing a methionine mimic, l-azidohomoalanine (l-AHA). Lysates were normalized by protein content, labeled with IRDye 800CW-DBCO, blotted on a nitrocellulose membrane, and imaged with a LI-COR Odyssey CLx Imager. (D) l- AHA bulk translation assay demonstrates that the degree of DNA damage influences the magnitude of translational repression. HEK cells were dosed with different concentrations of etoposide for 16 h, and the AHA assay was performed as detailed in (C) with the exception that samples were run on an SDS/PAGE gel. (E) eIF2a (S51) phosphorylation increases in HEK cells treated with 5 µm etoposide for 16 h. (F) eIF2a (S51) phosphorylation does not increase in HEK cells 72 h post Cas9-sgIntron electroporation. (G) Western blotting indicates that co-administration of 200 nm ISRIB with 5 µm etoposide does not rescueRPS27A 16 h post drug treatment. (H) 4E-BP1 (T37/47) phosphorylation does not change after dsDNA damage. Cells were either treated with 5 µm etoposide or electroporated with Cas9-sgIntron. Treatment with 2.5 µm PP242 for 30 min served as a positive control for 4E-BP1 hypo-phosphorylation.