Nonradioactive Labeling Research Articles

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies

Plain Language SummaryWe developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells’ platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband’s or patient’s own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.

Scale-Up of a Heck Alkenylation Reaction: Application to the Synthesis of an Amino-Modifier Nucleoside ‘Ruth Linker’

AbstractRuth linker is a C5 pyrimidine modified nucleoside analogue widely utilized for the incorporation of a primary amine in a synthetic oligonucleotide. The increasing demand for non-radioactive labeling, detection of biomolecules, and assembly of COVID-19 test kits has triggered a need for scale-up of Ruth linker. Herein, an efficient protocol involving a palladium-catalyzed Heck alkenylation is described. The synthesis has been optimized with a goal of low catalyst concentration, column-free isolation, high product purity, reproducibility, and shorter reaction time. The scalability and utility of the process have been demonstrated successfully on a 100 g scale (starting material). Additionally, for scale-up of the Heck alkenylation protocol, 7-phospha-1,3,5-triaza-adamantanebutane sulfonate (PTABS) as the coordinating caged phosphine ligand was also synthesized on a multigram scale after careful optimization of the conditions.

Assays for Autophagy III: Observing Dynamic Protein Trafficking.

Macroautophagy, by its very nature, is a protein trafficking process. Cargos are transported and processed. Atg proteins come and go. In this chapter, we present three assays to monitor these dynamic events: a non-radioactive pulse-chase labeling assay to monitor the transport of prApe1 and two fluorescent microscopy-based assays to assess the trafficking of Atg8 and Atg9.

Qualitative and quantitative evaluation of rice component in cereal products by the DNA-based technology

A method based upon the polymerase chain reaction (PCR) has been developed for distinguishing rice species from other cereals. Two specific primer sets designed from 5S ribosomal RNA genes and promoter of rice glutelin genes were able to identify even small amount of DNA fragments in rice. DNA isolated from grain, flour and such products gave similar results. Furthermore, evaluation of quantitative analysis of rice in the samples was accomplished by hybridization with probe designed from promoter of rice glutelin genes. The analysis of a lab-made sample (a mixture of TCW70 rice flour and cassava starch) with nonradioactive labeling probe was performed. The R2 of calibration curve was 0.98 and the calculated amount of rice flour in the sample was very close to the actual value with CV < 3% (n = 2). However, this method is only proper for rice flour, because it is difficult, but necessary, to select a suitable standard that can exhibit rice species and broken situation of DNA about the samples.

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos.

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.

In vivo analysis of protein translation activity in sea urchin eggs and embryos.

Protein synthesis is a major regulatory step of gene expression in different physiological processes including development. Translation of proteins in sea urchin is stimulated upon fertilization and is necessary for cell cycle progression and development. Translational control is exerted through multifactorial mechanisms, including mRNA recruitment into polysomes and increased rates of translational activity. In this chapter, we review the methods used in sea urchin eggs and embryos to analyze translation activity in vivo both from perspectives of the proteins and of the mRNAs. First, we describe methods to quantify or visualize newly synthesized proteins with radioactive and non-radioactive labeling techniques. Next we present the polysome isolation and profiling on sucrose gradients, allowing the identification of translated mRNAs. Finally, we outline a procedure to follow the translation of a reporter luciferase protein from an mRNA microinjected into the egg.

Sex-linked AFLP marker identification in dioecious Betelvine (Piper betle L.)

ABSTRACT This study aimed to develop sex-specific AFLP markers for dioecious Piper betle (Betelvine). A combination of ulk segregant analysis (BSA) and non-radioactive labelling led to simple, rapid and reliable PCR-based screening of sex-specific markers. A total of nine primer combinations were screened using BSA for male and female cultivars. Seven out of nine primer combinations produced bands specific to either of the sex. These seven primer combinations when used for screening DNA sample from individual male and female cultivars yielded two primer combinations which produced male sex-specific markers. Primer combinations EcoRI-CTA/MseI-CTA and EcoRI-CGT/MseI-CGT produced unique fragments of ~ 147 bp and ~ 326 bp, respectively, in bulk DNA as well as in DNA sample from individual male and female cultivars. These unique fragments were present in all the tested males and absent from all the tested female cultivars.

Uptake Pathways of Protein-Coated Magnetic Nanoparticles in Platelets.

Magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. Platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (HSA). However, the optimal HSA density coated on particles and the uptake mechanism of single particles in platelets remain unclear. Here, we utilized single-molecule force spectroscopy (SMFS) and other complementary methods to characterize the interaction of particles when interacting with platelets and to determine the optimal HSA amount required to coat particles. An HSA concentration of 0.5-1.0 mg/mL for coating particles is most efficient for platelet labeling. Binding pathways could be elucidated by linking a single HSA particle to SMFS tips via polyethylene glycol (PEG) linkers of different lengths and allowing them to interact with immobilized platelets on the substrate. Depending on the PEG length (i.e., short ∼2 nm, medium ∼30 nm, and long ∼100 nm), particles interact differently with platelets as shown by one, two, or three force distributions, which correspond up to three different binding pathways, respectively. We propose a model that the short PEG linker allows the particle to interact only with the platelet membrane, whereas the medium and long PEG linkers promote the particle to transfer from open canalicular system to another target inside platelets. Our study optimizes magnetic platelet labeling and provides details of particle pathways in platelets.

Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity.

Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.

Abstract 215: Tracking transplanted cells with paramagnetic fluorinated nanoemulsions

Abstract Visualization of distinct cell populations in vivo is one of the most formidable challenges in biomedical sciences. Clinical translation of cutting-edge therapies that involve administration of live immunotherapeutic or stem cells can benefit from understanding the fate of injected cells in vivo. Magnetic resonance imaging (MRI) is emerging as a prime method for non-invasive, longitudinal cell tracking. Fluorine-19 MRI (19F MRI) involves the use of non-radioactive 19F label in the form of biologically inert, cytocompatible perfluorocarbons (PFC). Immediately prior to injection, cells of interest are labeled ex vivo with water-based PFC nanoemulsions. 19F MRI yields background-free images of labeled cells in an anatomical context provided by conventional 1H MRI. Both datasets are acquired on the same scanner in one imaging session. This technology has recently been used to detect immunotherapeutic dendritic cells delivered to colorectal adenocarcinoma patients. We sought to enhance sensitivity and versatility of 19F MRI by using paramagnetic relaxation agents. Covalent modification of clinically relevant PFCs with metal-binding ligands enabled stable and uniform incorporation of gadolinium and iron in the fluorous phase of nanoemulsions. Paramagnetic relaxation enhancement resulted in up to 50-fold reduction in 19F longitudinal relaxation time (T1) compared to state-of-the-art commercial tracers. The ultralow 19F T1 of our paramagnetic tracers enabled in vivo cell tracking with rapid, three-dimensional, zero time-to-echo (ZTE) 19F MRI pulse sequences in mice bearing subcutaneous allografts of 19F-labeled GL261 cells. Moreover, the high local concentration of paramagnetic metals imparted negative contrast in T2*-weighted 1H images. Combination of the new agents with conventional 19F tracers comprises a previously unavailable toolbox of 19F contrast agents, selectively detectable by appropriate acquisition methods. This enables “multicolor” 19F MRI tracking of multiple cell populations, e.g., for monitoring the interaction of host immune cells with injected therapeutic cells or cancer xenografts. Overall, the design of biocompatible, fast-relaxing 19F tracers addresses the issue of modest sensitivity of 19F MRI and reduces the barriers to widespread adoption of this powerful imaging technique. Citation Format: Alexander A. Kislukhin, Hongyan Xu, Stephen R. Adams, Kazim Narsinh, Roger Y. Tsien, Eric T. Ahrens. Tracking transplanted cells with paramagnetic fluorinated nanoemulsions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2015-215

Fragmentation patterns of boron-dipyrromethene (BODIPY) dyes by electrospray ionization high-resolution tandem mass spectrometry.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. Unusual radical molecular cations ([M](+•)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+•) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra. Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.

A simple, sensitive and safe method to determine the human α/β-tryptase genotype.

The human tryptase locus on chromosome 16 contains one gene encoding only β-tryptase and another encoding either β-tryptase or the homologous α-tryptase, providing α:β gene ratios of 0∶4, 1∶3 or 2∶2 in the diploid genome, these genotypes being of potential clinical relevance in severe atopy. Using an EcoRV restriction site in α- but not β- tryptase, PCR products, spanning intron 1 to exon 5, were used to determine α/β-tryptase gene ratios using non-radioactive labels, including ethidium bromide labeling of all PCR products, and either digoxigenin-primer or DY682-primer labeling of only the final PCR cycle products. Sensitivity increased ∼60-fold with each final PCR cycle labeling technique. Ethidium bromide labeling underestimated amounts of α-tryptase, presumably because heteroduplexes of α/β-tryptase amplimers, formed during annealing, were EcoRV resistant. In contrast, both final PCR cycle labeling techniques precisely quantified these gene ratios, because only homoduplexes were labeled. Using the DY682-primer was most efficient, because PCR/EcoRV products could be analyzed directly in the gel; while digoxigenin-labeled products required transfer to a nitrocellulose membrane followed by immunoblotting. This technique for determining the α/β-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for α/β-tryptases, and for other closely related alleles.

A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells.

BackgroundWith the rapid advancement of cell biology, the evaluation of a given protein’s synthesis and release in cells becomes critical. However, up to now there has been no technique available to morphologically visualize and measure a newly synthesized protein in cells, nor can we measure the protein’s release from the cells.ResultsIn this study, we developed a set of assays combining pulse chase amino acid substitution, non-radioactive labeling, and immunofluorescence co-localization to visualize newly synthesized proteins in individual cells and then to detect their release using modified ELISA. We demonstrated the synthesis and release of Bcl-2, MMP-9, and immunoglobulin G (IgG) in a human trophoblast cell line, of which the last finding has not been reported previously.ConclusionsThis new technique offers a powerful tool to evaluate the dynamics of the synthesis and release of target proteins in individual cultured cells with wide applications in genetic and protein analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0045-1) contains supplementary material, which is available to authorized users.

Tools and Strategies to Match Peptide-Ligand Receptor Pairs.

Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.

LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.

Chemical modification of multiwalled carbon nanotube with a bifunctional caged ligand for radioactive labelling

Carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs) have been successfully radiolabelled with cobalt-57 (57Co) (T1/2=270days) via the attachment of the bifunctional caged ligand MeAMN3S3sar. In this study MeAMN3S3sar has been synthesized and coupled to MWCNTs to form the conjugate MWCNT–MeAMN3S3sar. Synthesis was confirmed with nuclear magnetic resonance. X-ray photoelectron spectroscopy (XPS) confirmed the conjugation. Non-radioactive labelling of this conjugate was completed with Cu(II) ions to confirm the stability of the MeAMN3S3sar after coupling with the MWCNTs. The complexation of the Cu(II) was also confirmed with XPS. Transmission electron microscopy was used to demonstrate that the coupling reaction had a negligible effect on the size and shape of the MWCNTs. Radiolabelling of the MWCNT–MeAMN3S3sar conjugate and pristine (untreated) MWCNTs (non-specific) with the gamma-emitting radioactive isotope 57Co were compared. The radiolabelling efficiency of the MWCNT–MeAMN3S3sar conjugate was significantly higher (95% vs. 0.1%) (P⩽0.001) than for the unconjugated pristine MWCNTs. This will allow for the potential tracking of nanoparticle movement in vitro and in vivo.

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

Application of a nonradioactive method of measuring protein synthesis in industrially relevant chinese hamster ovary cells

Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a "snapshot" to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells.

P90RSKs mediate the activation of ribosomal RNA synthesis by the hypertrophic agonist phenylephrine in adult cardiomyocytes

Cardiac hypertrophy involves the growth of heart muscle cells and is driven by faster protein synthesis which involves increased ribosome biogenesis. However, the signaling pathways that link hypertrophic stimuli to faster ribosome production remain to be identified. Here we have investigated the signaling pathways which promote ribosomal RNA synthesis in cardiomyocytes in response to hypertrophic stimulation. We employed a new non-radioactive labeling approach and show that the hypertrophic agent phenylephrine (PE) stimulates synthesis of 18S rRNA (made by RNA polymerase I) and 5S rRNA (produced by RNA polymerase III) in adult cardiomyocytes. In many settings, rRNA synthesis is driven by rapamycin-sensitive signaling through mammalian target of rapamycin complex 1 (mTORC1). However, the activation of rRNA synthesis by PE is not inhibited by rapamycin, indicating that its regulation involves other signaling pathways. PE stimulates MEK/ERK signaling in these cells. Inhibition of this pathway blocks the ability of PE to activate synthesis of 18S and 5S rRNA. Furthermore, BI-D1870, an inhibitor of the p90RSKs, protein kinases which are activated by ERK, blocks PE-activated rRNA synthesis, as did a second p90RSK inhibitor, SL0101. BI-D1870 also inhibits the PE-stimulated association of RNA polymerase I with the rRNA promoter. These findings show that signaling via MEK/ERK/p90RSK, not mTORC1, drives rRNA synthesis in adult cardiomyocytes undergoing hypertrophy. This is important both for our understanding of the mechanisms that control ribosome production and, potentially, for the management of cardiac hypertrophy.