PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase methods
Aim. Evidence-backed execution summary for PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase methods from PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase.
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mouse
Subject model for the experiment.
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PHD1 controls muscle mass in a cell-autonomous fashion
reagent used in the protocol.
- Use
- We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig...
PHD1 interacts with LRS and controls LRS stability
reagent used in the protocol.
- Use
- Although PHDs are mainly known for their hydroxylation dependent functions, it has been shown that they interact with other proteins to modulate their stability and/or activity via hydroxylation-independent mechanisms. To study whether PHD1 and LRS interact, we overexpressed both PHD1-flag and LRS-myc in HEK 293T c...
PHD1 interacts with LRS and controls LRS stability
reagent used in the protocol.
- Use
- Interestingly though, whereas the interaction between PHD1 and LRS was weak under normal culture conditions, it was further promoted when the enzymatic activity of PHD1 was inhibited upon treatment with the hypoxia mimetic, dimethyl 2-oxoglutarate (DMOG), or during amino acid starvation (Fig. ). Based on these...
Viral infections
reagent used in the protocol.
- Use
- For lentiviral infections HEK293T cells were transfected with 8 µg of each lentiviral vector and 5 µg of pmd2.G and pCMV8.9 lentivirus packaging plasmids using Lipofectamine 2000 (Invitrogen, 11668019). For retroviral infections, HEK293T cells were transfected with 10 µg of each retr...
Methods
reagent used in the protocol.
- Use
- HEK293T cells (DSMZ, ACC 635) were maintained in DMEM (ThermoFisher Scientific; 41966052) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific, 10270-106). Freshly isolated myogenic progenitors (MPs) were cultured in a 1:1 ratio of DMEM (ThermoFisher Scientific, 12320032) and Ham's F-10 (1&#...
Methods
reagent used in the protocol.
- Use
- Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µ...
Methods
reagent used in the protocol.
- Use
- Insulin stimulation: differentiated myotubes were serum starved for 16 h and 100 nM insulin was added to the medium for 5, 10, 20, and 30 min.
In vitro pull-down assay
reagent used in the protocol.
- Use
- HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for...
PHD1 is required for leucine mediated mTORC1 activation
mTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or amino acids,. To study whether loss Phd1 affects amino acid mediated activation of mTORC1 in muscle, we administered L-leucine (leucine), t...
- Use
- mTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or amino acids,. To study whether loss Phd1 affects amino acid mediated activation of mTORC1 in muscle, we administered L-leucine (leucine), t...
Methods
Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µ...
- Use
- Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µ...
In vitro hydroxylation assay
The potential ability of PHD1 to hydroxylate LRS was studied by determining the amount of radioactive 4-hydroxyproline formed in the substrate. Full-length cDNAs for human LRS and HIF1α (positive control) were translated in the presence of L-[2,3,4,5-3 H]-proline (85 mCi/mmol, Perkin Elmer) in a rab...
- Use
- The potential ability of PHD1 to hydroxylate LRS was studied by determining the amount of radioactive 4-hydroxyproline formed in the substrate. Full-length cDNAs for human LRS and HIF1α (positive control) were translated in the presence of L-[2,3,4,5-3 H]-proline (85 mCi/mmol, Perkin Elmer) in a rab...
Animals
Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and...
- Use
- Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and...
Experimental procedures
Muscle force measurements: WT and PHD1 KO mice were sacrificed and soleus was excised and placed in Krebs-Henseleit buffer (120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl 2, 1.2 mM KH2PO4, 2 mM MgSO4) supplemented with 1 × MEM amino acid mixture (Invitrogen, 1...
- Use
- Muscle force measurements: WT and PHD1 KO mice were sacrificed and soleus was excised and placed in Krebs-Henseleit buffer (120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl 2, 1.2 mM KH2PO4, 2 mM MgSO4) supplemented with 1 × MEM amino acid mixture (Invitrogen, 1...
Experimental procedures
MRI: body composition was measured in 12-14-week-old PHD1 KO mice after short term food withdrawal (4 h) using EchoMRI-100H (EchoMRI) according to the manufacturer's instructions.
- Use
- MRI: body composition was measured in 12-14-week-old PHD1 KO mice after short term food withdrawal (4 h) using EchoMRI-100H (EchoMRI) according to the manufacturer's instructions.
Immunohistochemistry and histology
Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody...
- Use
- Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody...
Immunohistochemistry and histology
LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after w...
- Use
- LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after w...
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PHD1 levels and LRS activity decline during aging
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed anabolic resistance, is impaired -. Several molecular mechanisms underlying anabolic resistance have been proposed, including reduced amino acid delivery and uptake into the muscle, but it is not known whether leucine sensing mechanisms are affected. Thus, to explore the relevance of our findings in a human setting, we compared PHD1 protein content in muscle samples from a small cohort where samples were obtained from healthy old ( n = 8, 4 women and 4 men, 72.6 ± 2.3 y (mean ± SEM)) vs. young ( n = 8, 4 women and 4 men, 26.1 ± 1.1 y) volunteers after an overnight fast. In this cohort, old people showed reduced activ...
PHD1 controls muscle mass in a cell-autonomous fashion
We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing...
Methods
Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µm cell strainers and a heterogeneous cell population was purified by FACS sorting or by serial preplating. For FACS, MPs were sorted based on positive alpha 7-integrin (1:100) and absence of Sca1, CD31, and CD45 (1:1000, see Supplementary Table ). For serial preplating, a freshly extracted heterogeneous cell population was preplated for 1.5 h at day 0, 4, and 10 after cell extraction. MPs were cultured in growth medium (see above) on dishes coated with Matrigel Basement Membrane Matrix (Corning, #356237, 1/25 dilution). When cells reached 80% confluency, growth...
Methods
Leucine stimulation: differentiated myotubes were starved for 1 h in low-glucose DMEM lacking all amino acids (US Biology, D9800-13), supplemented with 10% dialyzed FBS (dFBS) (ThermoFisher Scientific, 26400-044). Leucine was supplemented for 30 min in the indicated concentrations. For the RagA leucylation experiments, differentiated myotubes were incubated with DMEM containing all AA, except leucine (Sigma, 9443) + 10% FBS for 16 h supplemented with 0, 0.8, or 5 mM leucine.
In vitro pull-down assay
HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for 20 min. A total of 300 µg protein from the supernatant fraction was incubated with ANTI-FLAG ® M2 Affinity Gel (Sigma, A2220-1ML) at 4 °C overnight. After washing 3 times in lysis buffer supplemented with 50 mM NaCl, the precipitates were dissolved in laemmli buffer.
Animals
Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and contained from 5′ to 3′: a 5.3-kb EcoRV-HindIII fragment comprising exon 2 and the majority of intron 2 as 5′ homology arm, a 1.8-kb floxed neor cassette, a 2.5-kb HindIII-EcoRV fragment comprising exon 3 through 6, in which a third loxP site was introduced downstream of exon 4 along with a novel EcoRI site for genotyping purposes, an SV40 thymidine kinase expression cassette for negative selection purposes. The introduced third loxP site together with the loxP site located 3′ of the neor cassette thus flanked the Phd1 gene segment comprising exon 3...
Immunohistochemistry and histology
Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody cocktail was applied for 120 min for myosin heavy chain I (1:50), IIa (1:200) and b (1:100) (see Supplementary Table ) diluted in PBST + 10% goat serum. After washing 3 times for 5 min, a secondary antibody cocktail, diluted in PBST + 10% goat serum, was applied for goat anti-mouse Alexa Fluor 488,350,568 and wheat germ agglutinin Alexa fluor 647 (1:250) (ThermoFisher Scientific, see Supplementary Table ) for 60 min. Slides were mounted after a 3 × 5 min wash, sealed with glass cover slips and imag...
Immunohistochemistry and histology
LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after which they were rinsed with ice-cold (4 °C) PBS once and fixed for 5 min with 4% paraformaldehyde in PBS. The chamber wells were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 min. After rinsing twice with PBS, the wells were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody for LAMP2 and mTOR (1:200) in blocking buffer overnight at 4 °C (see Supplementary Table ). Subsequently, the wells were rinsed twice with blocking buffer and incubated with secondary antibodi...
Measurement outputs
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Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed an...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A two-way ANOVA design was used to assess the statistical significance of differences between mean values over phenotype and treatment. When appropriate, Tukey post hoc test was...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
MTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or a...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Growth factors as well as eccentric contractions activate mTORC1 mainly through the inhibition of its inhibitory TSC complex,, the latter being a direct target of HIF-dependen...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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inferred from protocolPreprocessing / cleaning
Aging is associated with a loss of muscle mass.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed an...; A two-way ANOVA design was used to assess the statistical significance of differences between mean values over phenotype and treatment. When appropriate, Tukey post hoc test was...; MTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or a...; Growth factors as well as eccentric contractions activate mTORC1 mainly through the inhibition of its inhibitory TSC complex,, the latter being a direct target of HIF-dependen....
from paperStatistical comparison
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed an...; A two-way ANOVA design was used to assess the statistical significance of differences between mean values over phenotype and treatment. When appropriate, Tukey post hoc test was...; mTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or a...; To study whether the blunted leucine mediated mTORC1 activation upon Phd1 deletion was driven by muscle-intrinsic factors, we decided to cross Phd1 fl/fl mice with human skeleta...
from paperReporting output
Report representative outputs alongside summary comparisons for Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed an..., A two-way ANOVA design was used to assess the statistical significance of differences between mean values over phenotype and treatment. When appropriate, Tukey post hoc test was..., MTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or a..., Growth factors as well as eccentric contractions activate mTORC1 mainly through the inhibition of its inhibitory TSC complex,, the latter being a direct target of HIF-dependen....
inferred from protocolStructured statistical methods
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed an...; A two-way ANOVA design was used to assess the statistical significance of differences between mean values over phenotype and treatment. When appropriate, Tukey post hoc test was...; mTORC1 represents a main regulatory hub in the control of muscle protein synthesis in response to many anabolic signals, such as growth factors, eccentric contractions, and/or a...; To study whether the blunted leucine mediated mTORC1 activation upon Phd1 deletion was driven by muscle-intrinsic factors, we decided to cross Phd1 fl/fl mice with human skeleta...
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Evidence quotes (8)
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed anabolic resistance, is impaired -. Several molecular mechanisms underlying anabolic resistance have been proposed, including reduced amino acid delivery and uptake into the muscle, but it is not known whether leucine sensing mechanisms are affected. Thus, to explore the relevance of our findings in a human setting, we compared PHD1 protein content in muscle samples from a small cohort where samples were obtained from healthy old ( n = 8, 4 women and 4 men, 72.6 ± 2.3 y (mean ± SEM)) vs. young ( n = 8, 4 women and 4 men, 26.1 ± 1.1 y) volunteers after an overnight fast. In this cohort, old people showed reduced activation of muscle protein synthesis in response to milk protein (containing a high dose of leucine) ingestion when compared to young subjects. We found that older muscle on average showed an almost 50% decrease in PHD1 levels (Fig. ). Lower PHD1 content was confirmed using immunofluorescent sta...
We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing 0.8 mM leucine), but consistent with our in vivo data, the response to insulin was preserved (Supplementary Fig. ).
Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µm cell strainers and a heterogeneous cell population was purified by FACS sorting or by serial preplating. For FACS, MPs were sorted based on positive alpha 7-integrin (1:100) and absence of Sca1, CD31, and CD45 (1:1000, see Supplementary Table ). For serial preplating, a freshly extracted heterogeneous cell population was preplated for 1.5 h at day 0, 4, and 10 after cell extraction. MPs were cultured in growth medium (see above) on dishes coated with Matrigel Basement Membrane Matrix (Corning, #356237, 1/25 dilution). When cells reached 80% confluency, growth medium was switched to differentiation medium containing low-glucose DMEM, 2% HS and 1% P/S. MPs were fully differentiated after 3 days.
Leucine stimulation: differentiated myotubes were starved for 1 h in low-glucose DMEM lacking all amino acids (US Biology, D9800-13), supplemented with 10% dialyzed FBS (dFBS) (ThermoFisher Scientific, 26400-044). Leucine was supplemented for 30 min in the indicated concentrations. For the RagA leucylation experiments, differentiated myotubes were incubated with DMEM containing all AA, except leucine (Sigma, 9443) + 10% FBS for 16 h supplemented with 0, 0.8, or 5 mM leucine.
HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for 20 min. A total of 300 µg protein from the supernatant fraction was incubated with ANTI-FLAG ® M2 Affinity Gel (Sigma, A2220-1ML) at 4 °C overnight. After washing 3 times in lysis buffer supplemented with 50 mM NaCl, the precipitates were dissolved in laemmli buffer.
Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and contained from 5′ to 3′: a 5.3-kb EcoRV-HindIII fragment comprising exon 2 and the majority of intron 2 as 5′ homology arm, a 1.8-kb floxed neor cassette, a 2.5-kb HindIII-EcoRV fragment comprising exon 3 through 6, in which a third loxP site was introduced downstream of exon 4 along with a novel EcoRI site for genotyping purposes, an SV40 thymidine kinase expression cassette for negative selection purposes. The introduced third loxP site together with the loxP site located 3′ of the neor cassette thus flanked the Phd1 gene segment comprising exon 3 and 4, which encode part of the catalytic domain conferring the prolyl hydroxylase activity. The construct was linearized with NotI and electroporated into G4 ES cells of 129SvEv/C57BL6 origin (kind gift from A. Nagy, Toronto). After positive-negative drug selection with 200 µg/ml...
Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody cocktail was applied for 120 min for myosin heavy chain I (1:50), IIa (1:200) and b (1:100) (see Supplementary Table ) diluted in PBST + 10% goat serum. After washing 3 times for 5 min, a secondary antibody cocktail, diluted in PBST + 10% goat serum, was applied for goat anti-mouse Alexa Fluor 488,350,568 and wheat germ agglutinin Alexa fluor 647 (1:250) (ThermoFisher Scientific, see Supplementary Table ) for 60 min. Slides were mounted after a 3 × 5 min wash, sealed with glass cover slips and imaged with a epifluorescent microscope (Zeiss Axio observer Z.1) at 10 ×. Fiber cross-sectional area was automatically determined with a Muscle J plugin for Image J software. Muscle fiber type was manually counted on tile scans. To determine the presence of centrally nucleated fibers, sections wer...
LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after which they were rinsed with ice-cold (4 °C) PBS once and fixed for 5 min with 4% paraformaldehyde in PBS. The chamber wells were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 min. After rinsing twice with PBS, the wells were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody for LAMP2 and mTOR (1:200) in blocking buffer overnight at 4 °C (see Supplementary Table ). Subsequently, the wells were rinsed twice with blocking buffer and incubated with secondary antibodies (diluted in blocking buffer 1:500) for one hour at room temperature in dark. Individual myotubes were imaged with 60 × confocal microscope (Olympus olympus fluoview FV 3000). LAMP2-mTOR colocalization was determined by ImageJ using a plugin described by Moser et al.. This analysis combines o...
Machine-readable layer
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"name": "PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase methods",
"description": "Evidence-backed execution summary for PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase methods from PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase.",
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"name": "PHD1 levels and LRS activity decline during aging",
"text": "Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed anabolic resistance, is impaired -. Several molecular mechanisms underlying anabolic resistance have been proposed, including reduced amino acid delivery and uptake into the muscle, but it is not known whether leucine sensing mechanisms are affected. Thus, to explore the relevance of our findings in a human setting, we compared PHD1 protein content in muscle samples from a small cohort where samples were obtained from healthy old ( n = 8, 4 women and 4 men, 72.6 ± 2.3 y (mean ± SEM)) vs. young ( n = 8, 4 women and 4 men, 26.1 ± 1.1 y) volunteers after an overnight fast. In this cohort, old people showed reduced activ..."
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"name": "PHD1 controls muscle mass in a cell-autonomous fashion",
"text": "We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing..."
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"text": "Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µm cell strainers and a heterogeneous cell population was purified by FACS sorting or by serial preplating. For FACS, MPs were sorted based on positive alpha 7-integrin (1:100) and absence of Sca1, CD31, and CD45 (1:1000, see Supplementary Table ). For serial preplating, a freshly extracted heterogeneous cell population was preplated for 1.5 h at day 0, 4, and 10 after cell extraction. MPs were cultured in growth medium (see above) on dishes coated with Matrigel Basement Membrane Matrix (Corning, #356237, 1/25 dilution). When cells reached 80% confluency, growth..."
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"text": "Leucine stimulation: differentiated myotubes were starved for 1 h in low-glucose DMEM lacking all amino acids (US Biology, D9800-13), supplemented with 10% dialyzed FBS (dFBS) (ThermoFisher Scientific, 26400-044). Leucine was supplemented for 30 min in the indicated concentrations. For the RagA leucylation experiments, differentiated myotubes were incubated with DMEM containing all AA, except leucine (Sigma, 9443) + 10% FBS for 16 h supplemented with 0, 0.8, or 5 mM leucine."
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"name": "In vitro pull-down assay",
"text": "HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for 20 min. A total of 300 µg protein from the supernatant fraction was incubated with ANTI-FLAG ® M2 Affinity Gel (Sigma, A2220-1ML) at 4 °C overnight. After washing 3 times in lysis buffer supplemented with 50 mM NaCl, the precipitates were dissolved in laemmli buffer."
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"text": "Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and contained from 5′ to 3′: a 5.3-kb EcoRV-HindIII fragment comprising exon 2 and the majority of intron 2 as 5′ homology arm, a 1.8-kb floxed neor cassette, a 2.5-kb HindIII-EcoRV fragment comprising exon 3 through 6, in which a third loxP site was introduced downstream of exon 4 along with a novel EcoRI site for genotyping purposes, an SV40 thymidine kinase expression cassette for negative selection purposes. The introduced third loxP site together with the loxP site located 3′ of the neor cassette thus flanked the Phd1 gene segment comprising exon 3..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Immunohistochemistry and histology",
"text": "Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody cocktail was applied for 120 min for myosin heavy chain I (1:50), IIa (1:200) and b (1:100) (see Supplementary Table ) diluted in PBST + 10% goat serum. After washing 3 times for 5 min, a secondary antibody cocktail, diluted in PBST + 10% goat serum, was applied for goat anti-mouse Alexa Fluor 488,350,568 and wheat germ agglutinin Alexa fluor 647 (1:250) (ThermoFisher Scientific, see Supplementary Table ) for 60 min. Slides were mounted after a 3 × 5 min wash, sealed with glass cover slips and imag..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Immunohistochemistry and histology",
"text": "LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after which they were rinsed with ice-cold (4 °C) PBS once and fixed for 5 min with 4% paraformaldehyde in PBS. The chamber wells were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 min. After rinsing twice with PBS, the wells were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody for LAMP2 and mTOR (1:200) in blocking buffer overnight at 4 °C (see Supplementary Table ). Subsequently, the wells were rinsed twice with blocking buffer and incubated with secondary antibodi..."
}
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"tool": [
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"name": "PHD1 is required for leucine mediated mTORC1 activation"
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"name": "In vitro hydroxylation assay"
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"name": "Methods"
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"name": "In vitro pull-down assay"
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"headline": "PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase",
"datePublished": "2020",
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"name": "Benoit Smeuninx"
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