51 research outputs found
Additional file 1: Figure S1. of Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome
Quantitation of Western blotting in Fig. 1a and b. Figure S2. Gene transcription and translation pattern of WT cells and ESCO2-Corrected cells. Figure S3. The boxplots display the show expression of mRNAs with 5’TOP sequences (a), PRTE sequences (b), and Babel genes (c). Figure S4. Motif and GO terms associated with genes with increased translational efficiency upon L-leucine treatment in RBS cells. Figure S5. mTORC2 show regulated genes do not show a coherent response to L-leucine. Figure S6. GTL2-regulated miRNAs are increased in RBS cells independent of L-Leu. Figure S7. Homeobox (HOX) gene expression is reduced in RBS cells. (ZIP 4.64 MB
L-leucine partially improved developmental deficiencies of ESCO2 depleted embryos in a TOR pathway-dependent manner.
<p>(A). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng) and immediately separated for D-Leu or L-Leu incubation (10 mM) for 2 d.p.f. L-Leu supplement partially rescued development of ESCO2-morphant embryos at a gross level. Scale bar = 200 µm. Animals were categorized as mildly affected, severely affected, or dead to further quantify the rescue. (B). The number of severely malformed and lethal embryos was quantified for ESCO2-depleted embryos in the presence of D-Leu or L-Leu supplement at 4 days post fertilization. A total of 73-115 embryos were quantified per condition (ESCO2-5mis with D-Leu (n = 73), ESCO2-MO with D-Leu (n = 114), ESCO2-5mis with L-Leu (n = 97), and ESCO2-MO with L-Leu (n = 115)). (C). Embryos (1–2 cells) were injected with ESCO2-MO (4 ng) and immediately transferred to L-Leu incubation at different concentrations. L-Leu supplement ameliorated the developmental defects of ESCO2 morphant embryos in a dosage-dependent manner. While the image is representative, about 20 embryos were analyzed per group. Bar = 200 µm. (D). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng) and immediately separated into D-Leu or L-Leu incubation (3 mM) for 24 h.p.f.. By Western blot analysis, L-Leu supplement partially rescued phosphorylation of S6 in the ESCO2-MO embryos. S6 and tubulin serve as loading controls. Each sample contains ∼100 embryos. (E). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng), and immediately separated into D-Leu or L-Leu incubation (10 mM) for 3 d.p.f., in the presence or absence of 200 nM rapamycin. While the image is representative, about 15 embryos were analyzed per group. Rapamycin curtails L-Leu rescue of ESCO2-morphants, and enhances malformation of ESCO2-depleted embryos. Bar = 200 µm. (F). WT and ESCO2 mutant embryos (1–2 cells) were incubated with 10 mM D-Leu or L-Leu for 4 d.p.f., and photographed. L-Leu treatment partially rescued development of ESCO2 mutant embryos. While the image is representative, about 10 embryos were analyzed per group. Bar = 200 µm. (G). WT and ESCO2 mutant embryos (1–2 cells) were incubated with 10 mM D-Leu or L-Leu for 2 d.p.f. Western blotting shows L-Leu treatment partially restored phosphorylation of S6 in ESCO2 mutant embryos, but p53 elevation persists. S6 and tubulin serve as loading controls.</p
Nucleolar organization was severely disrupted in the immortalized RBS cells.
<p>WT, RBS, and corrected RBS cells were immunostained with antibodies to the nucleolar components nucleolin and fibrillarin, and imaged with confocal microscopy. Scale bar = 10 µm. DNA was stained with DAPI. The average size (µm<sup>2</sup>) of individual nucleoli was measured in the fibrillarin stained sample using Image J software. About 20 cells were quantified for each sample.</p
Human RBS cells showed severely slow proliferation and decreased protein translation.
<p>(A). Immortalized WT, RBS, and corrected cells were seeded into 6-well plates with 0.5×10<sup>5</sup> cells/mL. After 8 days in culture, immortalized RBS cells showed very poor proliferation, compared with wild type (WT) and ESCO2-corrected (Cor) cells (P<0.01). Inhibition of p53, by Pifα (10 µM) incubation, partially rescued proliferation of RBS cells (P<0.05, 2-way ANOVA). (B). By FACScan analysis, RBS cells accumulated in G2/M phase. (C). Untransformed RBS and normal human skin fibroblasts (HSF) and amniotic fluid-derived cells (AFC) were grown in DMEM plus 15% FBS. <sup>3</sup>H-uridine (5 µCi) was incubated with 10<sup>6</sup> cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD<sub>260/280</sub>. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of <sup>3</sup>H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to normal cells was calculated using an unpaired t test. P = 0.0039, HSF ESCO2-Mut <i>vs</i> HSF ESCO2-WT; P = 0.000017, AFC ESCO2-Mut <i>vs</i> AFC ESCO2-WT. (D). Equal numbers of untransformed RBS and normal HSF and AFC cells were grown in DMEM plus 15% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of <sup>35</sup>S-methionine for 4 hrs. Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for <sup>35</sup>S radioactivity in a liquid scintillation spectrometer to determine the amount of <sup>35</sup>S-methionine incorporated into proteins. P = 0.00086, HSF ESCO2-Mut <i>vs</i> HSF ESCO2-WT; P = 0.0005, AFC ESCO2-Mut <i>vs</i> AFC ESCO2-WT.</p
L-leucine rescues many aspects of development in ESCO2-depleted embryos.
<p>(A). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng), and immediately separated into D-Leu or L-Leu incubation (10 mM). After 5 d.p.f., the embryos were stained with Alcian blue to detect cartilage development. Scale bar = 200 µm. While the image is representative, about 15 embryos were analyzed per group. (B). WT or ESCO2 mutant embryos were treated with D-Leu or L-Leu (10 mM). After 5 d.p.f., the embryos were stained with Alcian blue to detect cartilage development. While the image is representative, about 10 embryos were analyzed per group. L-leucine partially rescued head size and cartilage formation in (A) and (B). (C). Cranial development was quantified for the data in (A) using the sum of the pq (palatoquadrate) cartilage and mc (Meckel's cartilage) divided by cranial length, as indicated with lines in (B). The measurement was done on 3 embryos per group. P<0.01, ESCO2-MO with D-Leu treatment <i>vs</i> ESCO2-5mis with D-Leu treatment; P<0.05, ESCO2-MO with L-Leu treatment <i>vs</i> ESCO2-MO with D-Leu treatment. (D). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (2 ng), and immediately separated for D-Leu or L-Leu incubation (10 mM). After 2 d.p.f., the embryos were stained with acridine orange to detect apoptotic cells. Scale bar = 200 µm. (E). The number of apoptotic cells was quantified. P<0.0001, ESCO2-MO with D-Leu treatment <i>vs</i> ESCO2-5mis with D-Leu treatment; P = 0.0006, ESCO2-MO with L-Leu treatment <i>vs</i> ESCO2-MO with D-Leu treatment. (F). Embryos (1–2 cells) were treated as in (A). After 2 d.p.f., single cell suspensions of 10 embryos were generated in triplicate by mashing and filtering in PBS/10% NCS through cell strainers (100 µm, BD Falcon). Cells were resuspended in PBS/10% NCS and suspensions used in Caspase-Glo 3/7 Assays (Promega) according to the manufacturer's instructions. Luminescence was measured after 75 minutes on a multi-detection microplate reader. L-Leu treatment suppressed caspase 3/7 activation in the ESCO2-MO embryos. P<0.0001, ESCO2-5mis with D-Leu <i>vs</i> ESCO2-MO with D-Leu; P<0.0001, ESCO2-MO with L-Leu <i>vs</i> ESCO2-MO with D-Leu. (G). Embryos (1–2 cells) were treated as in (A). Embryo length was measured every day up to 4 d.p.f. with a mini-ruler under a Leica Stereoscope for ESCO2-MO and 5-mismatched controls. L-Leu treated ESCO2 morphants had a significantly longer body length compared with ESCO2 morphants treated with D-Leu (P<0.0001, 2-way ANOVA). The measurements were performed from head to tail for 10 embryos per group.</p
ESCO2 depletion in zebrafish is associated with an increase in phospho-H3 staining, and L-leucine partially rescues the increase.
<p>(A). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng), and immediately separated for D-Leu or L-Leu incubation (10 mM). At 24 h.p.f., cells were immunostained with anti-phospho-Histone H3 (pH3) antibody to detect mitotic cells. Scale bar = 200 µm. (B). The number of cells in mitosis was quantified for 5 embryos per group. P<0.0001, ESCO2-MO with D-Leu treatment <i>vs</i> ESCO2-5mis with D-Leu treatment; P<0.0001, ESCO2-MO with D-Leu treatment <i>vs</i> ESCO2-MO with L-Leu treatment. (C). A working model for the pathways involved in RBS is presented. Due to mutation in ESCO2, significant intracellular stress occurs due to defects in DNA replication, repair, and rDNA processes. This stress is detected by AMPK which can signal the activation of p53 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003857#pgen.1003857-Jones2" target="_blank">[115]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003857#pgen.1003857-Okoshi1" target="_blank">[116]</a> and the phosphorylation of TSC2. The phosphorylation of TSC2 will act to inhibit mTORC1 and downstream effectors such as 4EBP1, S6, and S6 kinase with the ultimate effect being the inhibition of translation. With the addition of L-leucine (green arrows), the leucyl tRNA synthetase will collaborate with the Rag GTPase to activate mTORC1, partially rescuing translation.</p
Reduced ESCO2 function is associated with mTOR inhibition, p53 activation, and dramatic developmental phenotypes in zebrafish.
<p>Total levels of S6, S6K1, and tubulin serve as loading controls. (A). Embryos were microinjected with 4 ng ESCO2-5mismatched morpholino (ESCO2-5mis) or ESCO2-ATG morpholino (ESCO2-MO) and photographed at 24 hours post fertilization (h.p.f.) and 3 days post fertilization (d.p.f.). Scale bar = 200 µm. (B). Embryos were microinjected with 1, 2, or 4 ng ESCO2-5mis or ESCO2-MO and photographed at 3 d.p.f. Scale bar = 200 µm. (C). Embryos were microinjected with 4 ng ESCO2-5mis or 1, 2, or 4 ng ESCO2-MO to test the effect of dosage on phophorylation of S6 by Western blot at 2 d.p.f. (D). ESCO2 morphant embryos (2 ng) show inhibition of the TOR pathway and accompanying activation of p53 at 24 h.p.f.. (E). ESCO2-transgenic mutant zebrafish embryos show gross developmental abnormalities compared with WT embryos at 24 h.p.f. and 3 d.p.f.. Bar = 200 µm. (F). ESCO2 mutant embryos show reduced S6 phosphorylation and upregulation of p53, Mdm2, and p27 by Western blot analysis.</p
ESCO2 mutation is associated with mTOR inhibition in RBS cells.
<p>(A–D). Phosphorylation of S6K1/S6 and 4EBP1-γ subunit ratio divided by α/β subunit was downregulated in the human immortalized RBS cells (A–B) and primary cells (C–D), as measured by Western blot analysis. The results are representative of three independent experiments. Total levels of S6, S6K1, and tubulin serve as loading controls. The relative ratios of protein expression were quantitated with ImageQuant TL software in (B, D). (E). L-Leu application partially improved poor proliferation of immortalized RBS cells. Each plot represents the average ± SEM of the ratio of the measurement of the indicated cell number, as calculated for three independent samples for 2-way ANOVA statistical analysis. P<0.01, HSF ESCO2-Mut with 10 mM D-Leu <i>vs</i> HSF ESCO2-WT with10 mM D-Leu; P<0.05, HSF ESCO2-Mut with 10 mM D-Leu <i>vs</i> HSF ESCO2-Mut with 10 mM L-Leu. (F). Elevated levels of cell death were partially suppressed by L-Leu supplementation. P<0.01, HSF ESCO2-Mut with 10 mM D-Leu <i>vs</i> HSF ESCO2-WT with 10 mM D-Leu; P<0.05, HSF ESCO2-Mut with 10 mM L-Leu <i>vs</i> HSF ESCO2-Mut with 10 mM D-Leu. (G). L-Leu, but not D-Leu, partially rescued the phosphorylated form of S6 in primary RBS fibroblasts. p53 levels were not rescued by treatment with L-Leu.</p
The morphology of AF cells, NP cells and PKH26-labeled NP cells and the proliferation of PKH26-labeled NP cells.
<p>(A) Optical microscopy of the morphology of AF cells. AF cells showed a long spindle-like morphology. (B) Optical microscopy of the morphology of NP cells. NP cells showed a short spindle-like or oval morphology. (C) Fluorescence microscopy of PKH26-labeled NP cells. The labeled cells displayed uniformly red fluorescence. (D) MTT assay of the proliferation of PKH26-labeled and-unlabeled NP cells. There was no significant difference in cell proliferation between PKH26-labeled and-unlabeled NP cells at the same culture time (P>0.05).</p
The histological staining of cell—scaffold constructs cultured for 6 weeks in dorsal subcutaneous pockets created in nude mice.
<p>(A-C) Safranin O staining of cross sections of cell—scaffold constructs. Safranin O was positive for both AF and NP phases, indicating the presence of ECM rich in sulfated proteoglycan. (D) Safranin O staining of cross sections of native pig disc, with both AF and NP positive. (E-G) Immunohistochemistry of collagen I in constructs. Immunohistochemistry of collagen I was intensively positive for both AF and NP phases. (H) Immunohistochemistry of collagen I in native pig disc. (I-K) Immunohistochemistry of collagen II in constructs. Immunohistochemistry of collagen II was positive for the NP phase and weakly positive for the AF phase. (L) Immunohistochemistry of collagen II in native pig disc. AF and NP phases of scaffold were closely connected after culture for 6 weeks <i>in vivo</i> (arrows indicating interface).</p
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