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. 2020 Mar 6;12(3):e9469.
doi: 10.15252/emmm.201809469. Epub 2020 Jan 31.

Inhibiting MARSs Reduces Hyperhomocysteinemia-Associated Neural Tube and Congenital Heart Defects

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Free PMC article

Inhibiting MARSs Reduces Hyperhomocysteinemia-Associated Neural Tube and Congenital Heart Defects

Xinyu Mei et al. EMBO Mol Med. .
Free PMC article

Abstract

Hyperhomocysteinemia is a common metabolic disorder that imposes major adverse health consequences. Reducing homocysteine levels, however, is not always effective against hyperhomocysteinemia-associated pathologies. Herein, we report the potential roles of methionyl-tRNA synthetase (MARS)-generated homocysteine signals in neural tube defects (NTDs) and congenital heart defects (CHDs). Increased copy numbers of MARS and/or MARS2 were detected in NTD and CHD patients. MARSs sense homocysteine and transmit its signal by inducing protein lysine (N)-homocysteinylation. Here, we identified hundreds of novel N-homocysteinylated proteins. N-homocysteinylation of superoxide dismutases (SOD1/2) provided new mechanistic insights for homocysteine-induced oxidative stress, apoptosis and Wnt signalling deregulation. Elevated MARS expression in developing and proliferating cells sensitizes them to the effects of homocysteine. Targeting MARSs using the homocysteine analogue acetyl homocysteine thioether (AHT) reversed MARS efficacy. AHT lowered NTD and CHD onsets in retinoic acid-induced and hyperhomocysteinemia-induced animal models without affecting homocysteine levels. We provide genetic and biochemical evidence to show that MARSs are previously overlooked genetic determinants and key pathological factors of hyperhomocysteinemia, and suggest that MARS inhibition represents an important medicinal approach for controlling hyperhomocysteinemia-associated diseases.

Keywords: N-homocysteinylation; acetyl homocysteine thioether; methionyl-tRNA synthetase; neural tube defects; reactive oxygen species.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. MARS expression levels were increased in samples with gains in copy numbers and developing/proliferating cells

MARS (A) and MARS2 (B) mRNA expression levels were measured by real‐time PCR in blood samples with 2 and 3–4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples.

Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3–4 copies. Protein‐level intensity shown in (D) was quantified using ImageJ and divided by the protein‐level intensity of actin (n = 6). The results were then graphed relative to samples with 2 copies.

MARS2 protein expression levels were detected (E) and quantified (F) (n = 6). Actin was used as a loading control.

Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co‐stained. Scale bar: 150 μm.

Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.

MARS expression was detected in E80 rhesus monkey brain sections (n = 3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinal layer, and blue indicates immature neurons. Scale bar: 1mm.

MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by Western blot. Each D/C lane pair was quantified relative to D as shown at the bottom. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n = 6).

MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n = 6).

MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n = 6).

MARS mRNA levels (n = 6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n = 6).

MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by Western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.

Representative result of MARS expression was detected by IHC in tumour tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.

Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.

Data information: Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used for (A, B, D and F). Wilcoxon matched pairs test was used for (K, L, M and N).Source data are available online for this figure.
Figure EV1
Figure EV1. Developing and proliferating cells have high MARS levels

Double IHC staining for MARS (green) and Ki67 (red) in brain sections at E8.5 and E9. Scale bar: 150 μm.

MARS2 expression was detected in human brain sections (n = 3) by IHC. Local large magnification is shown as framed by dashed lines. Red arrow indicates germinal layer, blue indicates immature neurons. Scale bar: 1 mm.

(Left) Proliferation markers Ki67 and MCM2 were double‐immunostained in coronal brain sections of the E80 monkey brain. (Right) Double‐stained MARS and MCM2. Scale bar: 1 mm.

Figure 2
Figure 2. MARSs potentiate Hcy‐induced apoptosis and growth inhibition

Schematic representation of MARS functions.

Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1‐Flag‐Bcl‐2 plasmids (n = 3). 5 μg plasmids were transfected in each 1 × 106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved‐caspase3 (c‐caspase3) levels were detected to determine apoptosis levels (right).

Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)‐ and MARS2 (D)‐over‐expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n = 4).

Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n = 4).

Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n = 4).

Metabolic characterization of untreated and MARS‐over‐expressing NE4C cells based on pathway analysis of significantly changed metabolites. Columns show significantly enriched pathways, and yellow columns indicate oxidative stress‐associated pathways.

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.Source data are available online for this figure.
Figure 3
Figure 3. MARS/MARS2 results of ROS accumulation by inducing N‐Hcy

HTL and ROS (superoxide) levels were compared (n = 4) between NE4C cells transfected with the empty vector, MARS (A) or MARS2 (B) plasmids. Data were normalized against that of vector‐transfected cells. HTL levels were determined by LC‐MS, and ROS levels were determined by DHE staining assay.

MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by Western blot. HTL and superoxide levels were quantified (n = 4) relative to vector‐transfected NE4C cells. Cells were cultured in 20 μM Hcy‐containing media.

Relative superoxide levels were determined by DHE staining assay and compared (n = 4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy‐containing media. MARS2 knockdown efficiencies were confirmed by Western blot.

NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n = 4).

The cellular ROS (superoxide) levels were determined (n = 4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.

Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS‐expressing and control cells were determined relative to the untreated NE4C cells (n = 4).

NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N‐Hcy levels in HTL‐treated and control cells were detected by Western blot.

Protein N‐Hcy levels in response to MARS knockdown (by shRNA) and over‐expression were detected by Western blot.

Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N‐Hcy (J) and superoxide (K) levels (n = 4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells.

Data information: Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.Source data are available online for this figure.
Figure EV2
Figure EV2. MARS regulates ROS and Wnt/β‐catenin signalling by modulating SOD N‐Hcy

Cellular superoxide levels were determined (n = 4) for NE4C cells treated with 20 μM HTL for different lengths of time. Superoxide levels were normalized to those of untreated NE4C cells.

Superoxide levels in NE4C cells (n = 3) were determined in the absence and presence of 20 μM Hcy in the culture media in control and MARS2‐over‐expressing cells (normalized to untreated cells).

N‐homocysteinylation levels and specific activities of recombinant SOD1 and SOD2 were determined (n = 3) with and without incubation with 20 μM Hcy in tubes (in vitro). SOD enzymes were separated from the reaction mixture following the treatment. The specific activities of the SOD1 and SOD2 mutants were normalized to those of untreated SOD1 and SOD2, respectively (n = 4).

Flag‐tagged SOD1 and SOD2 were each over‐expressed in untreated and MARS knockdown NE4C cells. After affinity purification, N‐homocysteinylation levels and the relative specific activities of purified SOD1 and SOD2 were determined relative to those in untreated NE4C cells (n = 4).

Flag‐tagged SOD2 was over‐expressed in untreated and MARS2 knockdown NE4C cells. After affinity purification, N‐homocysteinylation levels and the relative specific activities of purified SOD2 were determined (n = 4).

Untreated and MARS/MARS2 knockdown NE4C cells were treated with either homocysteine (20 μM) or HTL (10 μM), and the gene expression of c‐Myc and cyclin D1 was detected 6 h after the start of the respective treatment by real‐time PCR. Error bars indicate SEM (n = 4).

NRX‐Flag and DVL1‐Myc were co‐transfected in NE4C cells. Cells were treated with Hcy and NAC as indicated. The relative DVL1 levels that co‐immunoprecipitated with NRX‐Flag were determined and quantified (n = 3).

Levels of β‐catenin in response to Hcy and NAC treatment in NE4C cells were detected by Western blot (n = 3). Representative Western blots are shown. The average intensities of quantified bands in untreated groups were set at 100%.

Data Information: Data are presented as the means ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.
Figure 4
Figure 4. HTL inactivates SOD1 and SOD2 through N‐Hcy

Compartments of N‐Hcy substrates identified by proteomic survey were predicted by WoLF PSORT.

WebLogo visualization of the amino acids spanning N‐Hcy‐modified lysine sites.

SOD activities were down‐regulated by in vitro N‐homocysteinylation. Flag‐tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2 and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity‐purified and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N‐Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n = 4).

Upon switching N‐Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N‐Hcy unmodifiable mutants displayed lower specific activity than the wide‐type (WT) SOD. SOD1 (WT, K23W, K122W, K128W and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity‐purified SOD proteins was determined and normalized to protein levels. Wild‐type SOD1 and SOD2 activities were set at 100% (n = 4).

Hcy and HTL treatment in NE4C cells increased N‐homocysteinylation of SODs and decreased their activities. Flag‐tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum‐free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells (n = 4).

N‐Hcy SOD levels were down‐regulated in MARS/MARS2 knockdown cells. Flag‐tagged SOD1 and SOD2 were each over‐expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N‐Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n = 4).

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.Source data are available online for this figure.
Figure 5
Figure 5. N‐Hcy activates Wnt signalling through ROS‐induced disruption of DVL1‐NRX interactions

Levels of β‐catenin in response to Hcy treatment in NE4C cells were detected by Western blot (n = 3). Representative Western blots are shown, and band intensities were quantified relative to the untreated group.

Levels of β‐catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells (n = 4).

TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n = 4). TCF activities responding to either 20 μM Hcy‐ or 10 μM HTL‐treated were measured by quantifying Topflash/Fopflash relative to the untreated groups.

Levels of β‐catenin in untreated and MARS knockdown NE4C cells were detected (n = 3) with the presence or absence of 20 μM Hcy in the culture media.

NE4C cells were co‐transfected with NRX‐Flag and DVL1‐Myc plasmids and treated with different concentrations of Hcy (E) or HTL (F). The relative amount of DVL1 that co‐immunoprecipitated (co‐IPed) with NRX‐Flag was determined (n=3).

NRX‐Flag and DVL1‐Myc were co‐transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co‐IPed with NRX‐Flag were determined and quantified (n = 3) for treated and control cells cultured with or without 20 μM Hcy.

Flag‐tagged SOD1 and SOD2 were co‐over‐expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β‐catenin in cells with and without SOD1/2 expression were determined (n = 3). (I) Relative levels of DVL1 that co‐IPed with NRX‐Flag were quantified in SOD‐expressing and control cells cultured with or without 10 μM HTL (n = 3).

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.Source data are available online for this figure.
Figure 6
Figure 6. AHT inhibits Hcy sensing

Chemical structures of Hcy and its structural analogue acetyl Hcy thioether (AHT) are shown. The analogous parts are circled with dashed lines.

MARS‐ or MARS2‐catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.

MARS and MARS2 activities were determined (n = 4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy.

Relative HTL levels were determined (n = 4) for NE4C cells treated with various AHT concentrations for 4 h.

AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC‐MS.

Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments (n = 6).

NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N‐homocysteinylation levels were detected by Western blot. Band intensities were quantified relative to the untreated group using ImageJ (H) (n = 4).

Cellular superoxide levels were determined (n = 4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells.

Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT‐treated NE4C cells (n = 4).

Levels of β‐catenin in untreated, Hcy‐ or HTL‐treated NE4C cells were detected (n = 3) with the presence or absence of 70 μM AHT in the culture media. Mean β‐catenin levels in untreated NE4C cells were set at 100%.

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.Source data are available online for this figure.
Figure EV3
Figure EV3. AHT decreased protein N‐Hcy levels

Nuclear magnetic resonance (NMR) spectroscopic analysis was carried out to verify the success of synthesis. The 1H NMR (D2O) of AHT is shown: δ 3.94 (t, J = 8 Hz, 1 H), 3.40 (s, 2 H), 2.76 (t, J = 8 Hz, 2 H), 2.22–2.15 (m, 2 H) (Thomsen et al, 2013).

AHT (71 or 110 μg/kg per day) was injected in SD rats for 6 days. Relative HTL levels in liver homogenates were compared between AHT‐treated and untreated SD rats.

Plasma Hcy levels in AHT‐treated or untreated rats were determined by a biochemistry analyser (n = 4).

H9C2 cells were cultured in DMEM with 10 μM Hcy and supplemented the indicated AHT levels; N‐homocysteinylation levels were detected by Western blot. The data of N‐Hcy level quantification relative to the untreated group are shown in (E) (n = 4).

Superoxide levels in H9C2 cells (n = 4) were determined in the absence or presence of 10 μM Hcy in the culture media (normalized to untreated cells).

Data information: Data are presented as the means ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.
Figure EV4
Figure EV4. Targeting MARS to inhibit N‐Hcy decreased the ROS, β‐catenin and apoptosis levels

Hcy levels in maternal liver homogenates were detected in mice (A) and rats (B).

ATRA‐induced NTDs are exemplified by spina bifida (left) and exencephaly (right). Morphologies of E18.5 embryos from ATRA‐treated rats were examined. Scale bar: 3 mm.

Total protein N‐Hcy levels in maternal rat liver homogenates were determined by Western blot and quantified in correlation to NTD prevalence in the corresponding foetuses.

E10.5 foetal rats were subjected to DHE immunological staining to determine superoxide levels. Randomly selected foetal rats from litters of untreated, ATRA‐, ATRA + AHT‐ and ATRA + NAC‐treated pregnant rats were assayed (n = 3 each group). DHE fluorescence intensities are quantified at the bottom. Scale bar: 100 μm.

SOD1 and SOD2 were immunoaffinity purified from E10.5 foetal rat neural tube tissues. The neural tube homogenates of each group were mixed from 6 embryos. N‐Hcy levels and relative (to untreated) SOD activities were determined. Error bar indicates assay replicates (n = 4).

MARS (red) elevation in E8.5 spinal neural tubes of ATRA‐treated mice was associated with TUNEL (green, a) and β‐catenin elevation. AHT and NAC treatment down‐regulated TUNEL and β‐catenin levels without affecting MARS levels. Scale bar: 300 μm.

Quantification of TUNEL‐positive cells in experiment (G). Three sections from three embryos were pooled for analysis.

Quantification of β‐catenin levels in E8.5 spinal neural tubes in (G). The mean value of β‐catenin‐positive cells/total cell number was set at 100%. Three sections from three embryos were pooled for analysis.

IHC staining for β‐catenin (green) in E8.5 and E10 heart sections (n = 6). Scale bar: 300 μm (E8.5), 600 μm (E10).

Quantification of β‐catenin levels in developing hearts at E8.5 (K) and E10 (L) from (J) (n = 6).

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.
Figure 7
Figure 7. Hcy sensing inhibition by MARSs prevented NTD and CHD onset

Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N‐Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n = 3) and are presented in (B).

Double immunofluorescent staining (C) for MARS (green) and N‐Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n = 3) and are presented in (D).

Goss morphology of E10.5 ATRA‐treated and untreated mice embryos. The ATRA‐treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.

Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA‐treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1,000 μm.

Cardiac malformations were analysed by haematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA‐treated mice are as follows: ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1,000 μm

Total protein N‐Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N‐Hcy levels (ii).

Neural tube tissues at E10 were homogenized from litters of untreated, ATRA‐treated and ATRA + AHT co‐treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity‐purified from the homogenates. N‐Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n = 4).

Superoxide (a), apoptosis (b) and Wnt signalling (c, d) were over‐activated in ATRA‐treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole‐mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β‐catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).

Average (n = 3) DHE fluorescence intensities in experiment (J) (a) were quantified relative to the untreated group.

Quantification of TUNEL‐positive cells in experiment (J) (b). Three sections from three embryos were pooled for analysis.

Quantification of β‐catenin fluorescence intensities in experiment (J) (b) and (J) (c). The average (n = 3) β‐catenin level in the untreated group was set at 100%.

Hcy‐treated eggs exhibited NTD phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.

Total protein N‐Hcy levels of 72‐h chicken embryo homogenates from Hcy and Hcy + AHT‐treated groups were detected by Western blot.

Whole‐mount DHE staining of 72 h chicken embryos from untreated, Hcy‐, Hcy + AHT‐ and Hcy + NAC‐treated groups. Scale bar: 2000 μm.

Quantification of DHE fluorescence intensities in (P) (n = 3).

Data information: Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One‐way ANOVA with Dunnett's correction was used for multiple comparisons.Source data are available online for this figure.

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