Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Representative images of HE staining, Masson’s trichrome staining, TUNEL staining, and IHC staining for AARS1, lactylation, and H3K18la in renal biopsy samples from the participants in the present study (scale bar: 50 μm). The degree of damage to the kidney tissue structure, fibrosis, and kidney cell death, as well as the levels of AARS1, lactylation, and H3K18la, were increased in the kidneys of DN patients as the DN stage progressed. B Representative images of HE staining, Masson’s trichrome staining, TUNEL staining, and IHC staining for AARS1, lactylation, and H3K18la in renal biopsy samples from the control (Con) and DN model mice used in the present study (scale bar: 50 μm). The degree of damage to the kidney tissue structure, fibrosis, and kidney cell death, as well as the levels of AARS1, lactylation, and H3K18la, were increased in the kidneys of DN model mice. C Western blotting assays revealed that the protein levels of AARS1, lactylation, and H3K18la were increased in the kidneys of DN model mice. D qPCR assays indicated that the mRNA levels of AARS1 were increased in the kidneys of DN model mice. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Control, Western Blot

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Representative images of HE staining, Masson’s trichrome staining, TUNEL staining, and IHC staining for AARS1, lactylation, and H3K18la in renal biopsy samples from the control (Con), DN, AARS1 +/– , and AARS1 +/– DN model mice used in the present study (scale bar: 50 μm). Compared with DN model mice, AARS1 +/– DN model mice presented less damage to the kidney tissue structure, alleviated fibrosis, decreased kidney cell death, and reduced AARS1, lactylation, and H3K18la levels. B Compared with those in DN model mice, the protein levels of AARS1 and H3K18la were decreased in the kidneys of AARS1 +/– DN model mice. C Compared with those in DN model mice, the mRNA levels of AARS1 were decreased in the kidneys of AARS1 +/– DN model mice. D High-glucose treatment increased the levels of AARS1 and H3K18la, changes that were reversed by AARS1 silencing in HGECs and HK-2 cells. E High-glucose treatment increased the AARS1 mRNA level in HGECs and HK-2 cells, which was reversed by AARS1 silencing. F A PI assay indicated that high-glucose treatment increased cell death, which was reversed by AARS1 silencing in HGECs and HK-2 cells (scale bar: 100 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Control

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Molecular functions of the differentially expressed genes according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. B Representative images of IHC staining for 4-HNE, MDA, ACSL4, and GPX4 in renal biopsy samples from the participants in the present study (scale bar: 50 μm). IHC revealed that the levels of 4-HNE, MDA, and ACSL4 gradually increased, whereas the level of GPX4 gradually decreased in the kidneys of DN patients as the DN stage progressed. C Representative images of IHC staining for ACSL4 and GPX4 and transmission electron microscopy (TEM) images of renal biopsy samples from the control (Con), DN, AARS1 +/– , and AARS1 +/– DN model mice used in the present study (scale bar: 50 μm for IHC; scale bar: 500 nm for TEM). Compared with DN model mice, AARS1 +/– DN model mice presented reduced ACSL4 expression, upregulated GPX4 level,s and increased mitochondrial ridges and mitochondrial volumes. D Western blotting assays revealed that compared with DN model mice, AARS1 +/– DN model mice presented decreased ACSL4 levels and increased GPX4 expression in the kidney. E Compared with those in DN model mice, the MDA levels in the kidneys of AARS1 +/- DN model mice were decreased. F Compared with those in DN model mice, LPO levels were decreased in the kidneys of AARS1 +/– DN model mice. G Compared with that in DN mice, the Fe 2+ content was decreased in the kidneys of AARS1 +/– DN model mice. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Immunohistochemistry, Transmission Assay, Electron Microscopy, Control, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Western blotting assays indicated that AARS1 overexpression increased H3K18la, ELOVL5, and ACSL4 levels and decreased GPX4 levels in cells. Moreover, ELOVL5 silencing reversed the AARS1 overexpression-mediated increase in ACSL4 expression and decrease in GPX4 expression. B Results of the qPCR analysis of AARS1 and ELOVL5 levels in HGECs and HK-2 cells. C A C11-BODIPY 581/591 fluorescence probe was used to detect lipid peroxidation levels in HGECs and HK-2 cells. The results indicated that AARS1 overexpression increased lipid peroxidation levels, which was reversed by ELOVL5 silencing (scale bar: 100 μm). D A JC-1 fluorescence probe was used to detect changes in the mitochondrial membrane potential (MMP) of HGECs and HK-2 cells. Our results showed that the probes in the AARS1-overexpressing group were mainly green fluorescent monomers. In contrast, after the intervention with si-ELOVL5, the probes were converted into red fluorescent polymers, indicating that ELOVL5 silencing attenuated the destruction of the MMP in AARS1-overexpressing cells (scale bar: 100 μm). E MDA levels were elevated in AARS1-overexpressing cells, which were reversed via ELOVL5 silencing. F A FeRhoNox-1 fluorescent probe was used to detect the Fe 2+ content in the cells. Our results revealed that FeRhoNox-1 fluorescence was increased in AARS1-overexpressing cells but was decreased after ELOVL5 silencing (scale bar: 100 μm). G The red fluorescence intensity of MitoSOX, a mitochondria-specific superoxide indicator, was significantly increased in AARS1-overexpressing cells, but was reversed by ELOVL5 silencing (scale bar: 100 μm). H A PI assay indicated that AARS1 overexpression increased cell death, and this change was reversed by ELOVL5 silencing in HGECs and HK-2 cells (scale bar: 100 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Western Blot, Over Expression, Expressing, Fluorescence, Membrane

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A A mass spectrometry assay indicated that STAT1 may bind to AARS1. B The interaction between STAT1 and AARS1 in HGECs and HK-2 cells was verified by Co-IP. C STAT1 and AARS1 were colocalized in HGECs and HK-2 cells, as detected via an immunofluorescence assay (scale bar = 100 μm). D The direct interaction between STAT1 and AARS1 was verified by GST pull-down assays. E IHC data indicating that STAT1 levels in renal biopsy samples from diabetic nephropathy (DN) patients gradually increased as the DN stage progressed (scale bar: 50 μm). F Western blotting assays revealed that the STAT1 protein level was increased in the kidneys of DN model mice. G IHC data indicating that STAT1 levels are increased in the kidneys of DN model mice (scale bar: 50 μm). H qPCR revealed that the STAT1 mRNA level was increased in the kidneys of DN model mice. I qPCR assay results indicating that the STAT1 mRNA level was increased in hyperglycaemic cells. J Western blotting assays indicated that the STAT1 protein level was increased in hyperglycaemic cells. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Immunofluorescence, Western Blot

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Immunofluorescence assay results indicated that AARS1 overexpression promoted STAT1 nuclear translocation in HGECs and HK-2 cells. However, AARS1 5M did not have these effects (scale bar: 100 μm). B Western blotting assays indicated that AARS1 overexpression increased STAT1 phosphorylation in HGECs and HK-2 cells. However, AARS1 5M did not have these effects. C ChIP‒qPCR revealed that AARS1 overexpression enhanced the binding of STAT1 to the ELOVL5 promoter region in HGECs and HK-2 cells. However, AARS1 5M did not have these effects. D AARS1 lactylated STAT1 with lactate in vitro. E Co-IP assays indicated that AARS1 induced STAT1 lactylation in HGECs and HK-2 cells. Moreover, AARS1 overexpression increased STAT1, H3K18la and ELOVL5 levels in HGECs and HK-2 cells. However, AARS1 5M did not have these effects. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Immunofluorescence, Over Expression, Translocation Assay, Western Blot, Phospho-proteomics, Binding Assay, In Vitro, Co-Immunoprecipitation Assay

Journal: Cell Death and Differentiation

Article Title: AARS1-mediated lactylation of H3K18 and STAT1 promotes ferroptosis in diabetic nephropathy

doi: 10.1038/s41418-025-01587-4

Figure Lengend Snippet: A Representative images of HE staining, Masson’s trichrome staining, TUNEL staining, IHC staining for AARS1, H3K18la, STAT1, ELOVL5, ACSL4, and GPX4, and transmission electron microscopy (TEM) of renal biopsy samples from the control (Con), β-alanine, DN, and DN+β-alanine model mice (scale bar: 50 μm for HE, Masson, TUNEL, and IHC; scale bar: 500 nm for TEM). Compared with DN mice, DN+β-alanine mice presented less kidney tissue damage, fibrosis, and cell death; reduced AARS1, H3K18la, STAT1, ELOVL5, and ACSL4 expression; upregulated GPX4 levels; and increased mitochondrial ridges and volumes. B Western blotting assays revealed that compared with DN mice, DN+β-alanine mice presented decreased AARS1, H3K18la, STAT1, ELOVL5, and ACSL4 levels and increased GPX4 expression in kidney tissues. C qPCR assays indicated that compared with DN mice, DN+β-alanine mice presented decreased AARS1, STAT1 and ELOVL5 mRNA expression in kidney tissues. D Compared with those in DN model mice, the MDA levels were decreased in the kidneys of DN+β-alanine model mice. E Compared with those in DN mice, LPO levels were decreased in the kidneys of DN+β-alanine model mice. F Compared with that in DN mice, the Fe 2+ content was decreased in the kidneys of DN+β-alanine model mice. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: The AARS1 inhibitor Gln-AMS (HY-112861, MCE, China) was used in the cellular experiments.

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Transmission Assay, Electron Microscopy, Control, Expressing, Western Blot

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 1. G102R and R329H AARS1 are loss-of-function alleles that repress yeast cell growth in the presence of wild-type AARS1. (A) Yeast harboring an endogenous doxycycline-repressible ALA1 locus were transformed with a p413 vector with no insert and a pAG425 vector to express either wild-type or mutant human AARS1. Cultures were plated undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) Similar experiment as shown in panel A; here, yeast were first transformed with a p413 vector expressing wild-type human AARS1. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left side of the image, and the media conditions are indicated across the bottom of the image (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). Representative images are shown from thirteen (for G102R) or sixteen (for R329H) biological replicates. A cartoon on the bottom left illustrates the experimental conditions for all samples.

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Expressing, Cell Culture

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 2. G102R and R329H AARS1 dimerize with wild-type AARS1. (A) HEK293T cells were transfected with vectors to co-express wild-type and mutant human AARS1, and a western blot was performed to detect the resulting proteins along with endogenous loading controls. The image is representative of three independent replicates. A cartoon along the top illustrates the constructs employed in the experiments, and the presence or absence of each construct is indicated across the top of the gel image. Protein molecular weights are indicated in kilodaltons (kDa) along the left side of the image and antibodies are indicated along the right side. (B) After immunoprecipitation with an anti-6xHis antibody, a western blot was performed to detect co-immunoprecipitated proteins. A representative image from five (for R329H) or three (for G102R) independent replicates are shown. This image is annotated as in panel A. (C) After immunoprecipitation with an anti-FLAG antibody, a western blot was performed to detect co-immunoprecipitated proteins. A representative image from two independent replicates is shown. This image is annotated as in panels A and B. (D) After treating patient and control samples with a protein cross-linking agent, a western blot was performed to detect endogenous AARS1 protein. The image is representative of four independent technical replicates. Samples and conditions are indicated across the top of the image, protein molecular weights are indicated in kilodaltons (kDa) along the left side, and the antibody employed is indicated on the right side. DSS = disuccinimidyl suberate. (E) The percentage of dimeric AARS1 protein signal in the total AARS1 protein signal (D) was quantified with ImageJ. The mean of four technical replicates is shown, with error bars representing one standard deviation. A one-way ANOVA with Tukey’s multiple comparisons tests [F(2,9) = 0.3602, P = 0.7071] was performed to determine if there was a statistically significant difference between R329H/+ cells and either of the two controls.

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transfection, Mutagenesis, Western Blot, Construct, Immunoprecipitation, Control, Standard Deviation

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 3. Engineering dimer-reducing AARS1 variants. (A) A cartoon generated from PyMOL illustrates the crystal structure of the AARS1 C-terminal dimerization domain (left side). One subunit from the dimer is shown in green, and the other in purple. Amino-acid residues that contact the opposite subunit are shown in dark green or dark purple. The residues targeted in this assay are shown in pink and labeled in the inset. On the right side of this panel is a second cartoon that illustrates the AARS1 C-terminal dimerization domain with the Q855∗mutation. The dashed circles indicate the globular domain that is ablated by the premature stop codon. (B) Yeast harboring a doxycycline-repressible endogenous ALA1 locus were transformed with a pAG425 vector to express either wild-type or mutant human AARS1 (i.e. one of the engineered mutations affecting the residues highlighted in panel A). Cultures were plated undiluted or diluted on media lacking leucine, and containing galactose/raffinose and doxycycline. A representative image of four biological replicates is shown. The dilution of the spotted yeast cultured is indicated on the left and the media conditions are indicated across the bottom (leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). (C) Yeast protein lysates were subjected to western blot analysis to detect the human AARS1 proteins expressed from wild-type and mutant expression constructs, which are indicated across the top. Yeast was grown in galactose and raffinose media lacking leucine, with no doxycycline. A representative image of three biological replicates is shown.

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Generated, Labeling, Transformation Assay, Plasmid Preparation, Mutagenesis, Cell Culture, Western Blot, Expressing, Construct

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 4. Q855∗AARS1 impairs dimerization with wild-type AARS1. (A) HEK293T cells were transfected with vectors to co-express wild-type or Q855∗

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transfection

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 5. Reducing dimerization of G102R and R329H with wild-type AARS1 rescues yeast growth. (A) Yeast with the doxycycline-repressible endogenous ALA1 locus was transformed with an empty p413 vector and a pAG425 vector expressing wild-type or mutant human AARS1. Cultures were plated undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) Similar to strains shown in panel A, except that yeast was transformed with p413 expressing wild-type human AARS1. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left, and the media conditions are indicated across the bottom (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). (C) Yeast spot intensity was quantified using ImageJ. Bars represent the mean and one standard deviation. Thirteen biological replicates were assessed for G757∗and wild-type AARS1, eight for R329H and R329H + Q855∗, and seven for G102R and G102R + Q855∗. The indicated fold-change between the G102R strain and the G102R + Q855∗strain, and the fold-change between the R329H strain and the R329H + Q855∗strain, were both calculated using the mean of each sample. To compare yeast growth to the strain expressing both wild-type and G757∗AARS1, a one-way ANOVA with Dunnett’s multiple comparisons test was performed [F(5,50) = 19.90, P < 0.0001].

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Cell Culture, Standard Deviation

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 6. R329C, R329S and R326W AARS1 are loss-of-function alleles that dominantly repress yeast cell growth. (A) Yeast with the doxycycline- repressible endogenous ALA1 locus was transformed with a p413 vector with no insert, and a pAG425 vector expressing wild-type or mutant AARS1. Yeast cultures were spotted undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) A similar experiment to that described in panel A, except that yeast expresses wild-type human AARS1 from p413. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left, and the media conditions are shown across the bottom (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). Images are representative of three replicates.

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Cell Culture

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 7. Reducing dimerization of R329C, R329S or R326W with wild-type AARS1 rescues yeast growth. (A) Yeast with the doxycycline-repressible ALA1 locus was transformed with an empty p413 vector and a pAG425 vector expressing either wild-type or mutant AARS1. Yeast was spotted undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) An experiment similar to that described in panel A, except that yeast expresses wild-type human AARS1 from the p413 vector. (C) Yeast spot intensity was quantified using ImageJ analysis; bars represent the mean and one standard deviation. At least three biological replicates were assessed for all variants. The indicated fold change between the strains expressing R329C, R329S or R326W, and their counterpart with Q855∗in cis, was calculated using the mean intensity of each condition. To compare yeast growth to that of the strain expressing both wild-type and G757∗AARS1, a one-way ANOVA with Dunnett’s multiple comparisons test was performed [F(7,27) = 14.72, P < 0.0001].

Article Snippet: The Gateway cloning (Invitrogen) LR reaction was used to recombine the wild-type or mutant AARS1 locus into pAG425GAL-ccdB (Addgene #14153).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Standard Deviation