Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a The level of circulating ARMH4 decreased in aged people compared with young adults detected by mass spectrometry . Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent subjects. b The analysis of the public GTEx database about ARMH4 expression in blood samples from individuals aged 70-79 years compared to those aged 20-29 years. Young group are presented as box plots showing the median (0.0123), the 25th (0.0080) and 75th (0.0199) percentiles, and the minimum (0.0000) and maximum (0.0749) values, n = 69 (20–29); Old group are presented as box plots showing the median (0.0096), the 25th (0.0000) and 75th (0.0138) percentiles, and the minimum (0.0000) and maximum (0.0266) values, n = 26 (70–79). c The protein levels of Armh4 in the brain, lung, heart, liver, kidney and muscle from young (2–month-old) and aged (25-month-old) mice measured by western blot. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. d Schematic diagram illustrating the Armh4 gene knockout (KO) strategy in mouse. e Validation of Armh4 KO at the mRNA level in mouse tissues using qRT-PCR. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. f Mendelian inheritance of the breeding offspring from heterozygous Armh4 -KO mice. g Representative images showing fur appearance of wild type (WT) and Armh4 -KO mice at 2 months (young) or 25 months (aged) of age. Scale bar, 1 cm. h Weights of WT and Armh4 -KO mice during natural aging. Data are presented as mean values +/- SEM, with sample sizes of n = 12/9, 11/9, and 8/9 (WT/KO) for the 6–15, 18–21, and 24-month groups, respectively. i Representative Masson’s trichrome staining images showing the infrastructure of skins from young/aged WT and Armh4 -KO mice. n = 3. Scale bar, 200 μm. j Representative images showing the hair regeneration capacity after excising a patch of dorsal fur from 21-month-old WT or Armh4 -KO mice. n = 3. Scale bar, 1 cm. k , l Survival curves of two panels of WT and Armh4 -KO mice under natural feeding conditions. The mice from the first panel ( k ) were euthanized at 25 months of age for molecular studies, while mice from the second panel ( l ) were further raised until natural death. Statistical significance was determined by a two-tailed unpaired t -test, and the exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Mass Spectrometry, Expressing, Western Blot, Gene Knockout, Biomarker Discovery, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Body weight measurements of young (2-month-old) and aged (25-month-old) male WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 9 (WT young), 6 (KO young), 7 (WT aged), and 7 (KO aged) independent mice. b Organ weight measurements of young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 9 (WT young), 6 (KO young), 7 (WT aged), and 7 (KO aged) independent mice. c Representative liver images from young and aged WT and Armh4 -KO mice. Scale bar, 5 mm. d H&E staining of the liver and statistical analysis of inflammatory cell infiltration in young and aged WT and Armh4 -KO mice. Scale bar, 50 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 18 fields from 3 independent mice per group. e Liver Masson staining and statistical results in young and aged WT and Armh4 -KO mice. Scale bar, 50 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 15 fields from 3 independent mice per group. f Liver fibrosis index qRT-PCR results in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. g Liver β-gal staining and statistical results in young and aged WT and Armh4 -KO mice. Scale bar, 200 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 15 fields from 3 independent mice per group. h qRT-PCR results for liver aging and inflammation markers in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. i Blood glucose levels during GTT in 21-month-old mice. n = 10 (WT), 9 (KO). j qRT-PCR results for liver glucose metabolism markers in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. k qRT-PCR results for liver metabolic function markers in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. l qRT-PCR results for liver fatty acid metabolism markers in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. m–q ELISA quantification of serum metabolic profiles in young and aged WT versus Armh4 -KO mice at 8-week (young) or 25-month (aged) old: ( m ) Triglycerides (TG), ( n ) total cholesterol (TCh), ( o ) high-density lipoprotein cholesterol (HDL-C), ( p ) low-density lipoprotein cholesterol (LDL-C), ( q ) AST/ALT. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 (young groups) and n = 6 (aged groups) independent mice. Statistical significance was determined by two-way ANOVA, and the exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Representative echocardiographic images parasternal short-axis view (SAX) at the papillary muscle level from young and aged WT and Armh4 -KO mice heart. b , c The impacts of Armh4 deficiency on ejection fraction (EF%) ( b ) and fractional shortening (FS%) ( c ). Data are presented as mean values +/- SEM. with sample sizes of n = 12/9, 11/9, and 8/9 (WT/KO) for the 6-15, 18-21, and 24-month groups, respectively. Statistical significance was determined by a two-tailed unpaired t -test. d Representative echocardiographic images on the apical four-chamber view (A4C) from WT and Armh4 -KO mice (male and female) heart, and mitral E/A ratio was measured. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 16 (WT) and n = 10 (KO) independent mice at 15 months of age. Statistical significance was determined by a two-tailed unpaired t -test. e The impacts of Armh4 deficiency on left ventricle (LV) mass. Data are presented as mean values +/- SEM, with sample sizes of n = 12/9, 11/9, and 8/9 (WT/KO) for the 15, 18-21, and 24-month groups, respectively. Statistical significance was determined by a two-tailed unpaired t -test. f Representative heart images from young and aged WT and Armh4 -KO mice. Scale bar, 5 mm. g H&E staining of the heart in young and aged WT and Armh4 -KO mice. Scale bar, 50 μm. h WGA staining of the heart and statistical results of cardiomyocyte area in young and aged WT and Armh4 -KO mice. Scale bar, 50 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 24 fields from 3 independent mice per group. Statistical significance was determined by two-way ANOVA. i Heart masson staining and statistical results in young and aged WT and Armh4 -KO mice. Scale bar, 50 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 15 fields from 3 independent mice per group. Statistical significance was determined by two-way ANOVA. j qRT-PCR results of cardiac hypertrophy-related genes in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. Statistical significance was determined by two-way ANOVA. k qRT-PCR results of cardiac SASP-related genes in young and aged WT and Armh4 -KO mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 5 independent mice. Statistical significance was calculated with two-way ANOVA. l , PCA analysis results of cardiac RNA-seq from young and aged WT and Armh4 -KO mice, with sample sizes of n = 4 independent mice. m , Venn diagram analysis of differential genes from young and aged WT and Armh4 -KO mice, with sample sizes of n = 4 independent mice. n , Heatmap analysis of differentially expressed genes across WT-young, WT-aged, and Armh4 -KO groups, with sample sizes of n = 4 independent mice. o , Ericher analysis of differential expressed genes between WT and Armh4 -KO aged mice, with sample sizes of n = 4 independent mice. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Two Tailed Test, Staining, Quantitative RT-PCR, RNA Sequencing

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Validation of siRNA-mediated Armh4 knockdown efficiency in AC16 cells. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA. b Quantification of SA-β-gal cells in H 2 O 2 (400 μM)-induced senescence model of AC16 cells following Armh4 knockdown. Scale bar, 200 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for 12 fields from 3 biological replicates. Statistical significance was determined by two-way ANOVA. c Validation of Armh4 overexpression efficiency via plasmid transfection in AC16 cells. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA. d Quantification of SA-β-gal cells in H 2 O 2 (400 μM)-induced senescence model of AC16 cells following Armh4 overexpression. Scale bar, 200 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for 12 fields from 3 biological replicates. Statistical significance was determined by two-way ANOVA. e qRT-PCR results of SASP-related genes and Armh4 in WT, HET and KO MEFs. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was calculated with one-way ANOVA. f Validation of siRNA-mediated Armh4 knockdown efficiency in HUVECs. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA. g Quantification of SA-β-gal cells in D-Gal (10 mg/mL)-induced senescence model of HUVECs following Armh4 knockdown. Scale bar, 200 μm. Graph bars represent the mean percentage of SA-β-gal-positive area in Armh4 -knockdown cells treated with PBS or D-Gal, relative to their respective negative controls ± SEM. Black circles represent individual data points for 9 fields from 3 biological replicates. Statistical significance was determined by two-way ANOVA. h Validation of Armh4 overexpression efficiency via plasmid transfection in HUVECs. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA. i Quantification of SA-β-gal cells in D-Gal (10 mg/mL)-induced senescence model of HUVECs following Armh4 overexpression. Scale bar, 200 μm. Data are presented as mean values +/- SEM. Black circles represent individual data points for 9 fields from 3 biological replicates. Statistical significance was determined by two-way ANOVA. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Biomarker Discovery, Knockdown, Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Puromycin incorporation assay of WT and Armh4 -deficient MEFs to assess the effect on protein synthesis. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. b Puromycin incorporation assay of NRVMs with/without siRNA-mediated knockdown of Armh4 or adenoviral overexpression of Armh4 . Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. c Puromycin incorporation assay of NRVMs with/without exogenous administration of recombinant ARMH4. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. d Western blot analysis of autophagy-related markers in WT and Armh4 -deficient MEFs. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. e Immunofluorescence analysis using GFP-LC3-RFP plasmid labeling in WT and Armh4 -deficient MEFs to evaluate the impact of Armh4 knockout on autophagy. Scale bar, 20 μm. f , g Western blot analysis for autophagy-related markers p62 and LC3 in NRVMs with/without siRNA-mediated knockdown of Armh4 ( f ) or adenoviral overexpression of Armh4 ( g ). Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. h Western blot analysis for autophagy-related markers p62 and LC3 in NRVMs treated with/without recombinant ARMH4. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test, and the exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Knockdown, Over Expression, Recombinant, Western Blot, Immunofluorescence, Plasmid Preparation, Labeling, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Western blot analysis of phosphorylated p70S6K, Akt, ERK, and LC3 in the hearts of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. b Western blot analysis of phosphorylated, p70S6K, Akt, ERK, and LC3 in the livers of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. c Western blot analysis of phosphorylated mTOR, p70S6K, Akt, ERK in Armh4 -deficient MEFs compared to the WT controls. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. d , e Western blot analysis of phosphorylated mTOR, p70S6K, Akt, and ERK in NRVMs with/without siRNA-mediated knockdown of Armh4 ( d ) and adenoviral overexpression of Armh4 ( e ). Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. f Western blot analysis of phosphorylated p70S6K, Akt, and ERK in NRVMs treated with recombinant ARMH4. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Western Blot, Two Tailed Test, Knockdown, Over Expression, Recombinant

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Schematic diagram of IGF1R and FGFR1 activation pathways. b , c Co-IP ( b ) and immunofluorescence ( c ) to detect the interaction between ARMH4 and IGF1R/FGFR1 in HEK293 cells co-transfected with pcDNA3.1-h Armh4 -3FLAG and pcDNA3.1-h Igf1r -HA, as well as pcDNA3.1-h Armh4 -3FLAG and pcDNA3.1-h Fgfr1 -HA, Scale bar, 10 μm. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. d , e Validation of ARMH4-IGF1R ( d ) and ARMH4-FGFR1 ( e ) interaction using endogenous IP in heart tissues. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. f AlphaFold 3 simulation of the effect of ARMH4 on ligand-receptor binding for IGF1/IGF1R and FGF2/FGFR1. g AlphaFold3 simulation of the specific binding region of ARMH4 within the IGF1/IGF1R ligand-receptor complex. h Schematic diagram of the designed plasmid construct targeting the predicted binding sequence between ARMH4 and IGF1R. i Co-IP analysis of the interaction between IGF1R and ARMH4, including its isoforms, in HEK293 cells. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, Biomarker Discovery, Binding Assay, Plasmid Preparation, Construct, Sequencing

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a , b Adenoviral overexpression of Armh4 with PPP ( a ) and FA ( b ) treatment in NRVMs followed by Western blot analysis of phosphorylated mTOR, p70S6K, Akt and ERK. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. c , d Adenoviral overexpression of Armh4 with PPP ( c ) and FA ( d ) treatment in NRVMs followed by western blot to detect the autophagy-related markers. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. e , f Adenoviral overexpression of Armh4 with siRNA-mediated knockdown of Igf1r ( e ) and Fgfr1 ( f ) in NRVMs followed by western blot analysis of phosphorylated p70S6K, Akt, ERK and LC3. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. g , h Adenoviral overexpression of Armh4 with IGF1 ( g ) and FGF2 ( h ) treatment in NRVMs followed by western blot analysis of phosphorylated p70S6K, Akt, ERK and LC3. n = 3. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, and the exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Over Expression, Western Blot, Knockdown

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Western blot analysis of FGFR1, IGFR1 protein expression in the hearts of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. b Western blot analysis of FGFR1, IGFR1 protein expression in the livers of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. c Western blot analysis of FGFR1, IGFR1 protein expression in Armh4 -deficient MEFs. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. d, e Western blot analysis of FGFR1 and IGFR1 protein expression in NRVMs with/without siRNA-mediated knockdown of Armh4 ( d ) and adenoviral overexpression of Armh4 ( e ). Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. f Western blot analysis of FGFR1 and IGFR1 protein expression in NRVMs treated with recombinant ARMH4. n = 6. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Western Blot, Expressing, Two Tailed Test, Knockdown, Over Expression, Recombinant

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a TF-Target Finder predicted the transcription factors that co-regulated by IGF1R and FGFR1 screening diagram. b Western blot analysis of c-Myc protein expression in NRVMs upon Armh4 knockdown and overexpression. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. c, d qRT-PCR of Myc expression in NRVMs upon Armh4 knockdown ( c ) and overexpression ( d ). Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. e Schematic diagram of the dual-luciferase reporter constructs. f Dual-luciferase reporter assay in HEK293 cells. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. g Screening the c-Myc binding motifs by JASPAR. h Computational prediction of c-Myc binding motifs in IGF1R/FGFR1 regulatory regions using JASPAR. i Western blot analysis of c-Myc, IGF1R, FGFR1 expression in NRVMs upon Myc knockdown by siRNA. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. j–m Western blot analysis of IGF1R and FGFR1 expression ( j ), phosphorylated p70S6K, Akt, ERK ( k ), puromycin incorporation ( l ), the autophagy-related marker ( m ) in NRVMs with Myc knockdown followed by Armh4 overexpression. Representative blot of n = 4 biological replicates ( j , k , m ) and n = 3 biological replicates ( l ). Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 4 biological replicates ( j , k , m ) and n = 3 biological replicates ( l ). Statistical significance was determined by two-way ANOVA. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Armh4 -3FLAG, pcDNA3.1-m Armh4 -3FLAG and pcDNA3.1-r Armh4 -3FLAG recombinant plasmids were constructed by cloning the coding region of h Armh4 , m Armh4 and r Armh4 into the NheI (NEB, #R3131) and XhoI (NEB, #R0146) sites of the pcDNA3.1-3FLAG plasmid.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Two Tailed Test, Quantitative RT-PCR, Luciferase, Construct, Reporter Assay, Binding Assay, Marker

Journal: PLOS Biology

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1371/journal.pbio.3003163

Figure Lengend Snippet: (A) Experimental design: 10–12-week-old mice were treated with tamoxifen for five consecutive days (open arrowheads) during the first week of high-fat diet (HFD) feeding. After 12 weeks of HFD, Ang-II osmotic minipumps were implanted for 4 weeks; control mice were operated without minipump implantation. Maximum aortic diameter and BP were measured (Eco-BP) at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( B ) Representative ultrasound images of AbAo from mice before and after 4 weeks of Ang-II treatment. Yellow lines mark the lumen boundary. Scale bar, 1 mm. ( C, D ) AbAo diameter in (C) 7 Cn-Ctl and 8 EC-Cn −/− control-treated mice and 16 Cn-Ctl and 10 EC-Cn −/− Ang-II-treated, and in (D) 8 Cn-Ctl and 6 SM-Cn −/− control-treated mice and 18 Cn-Ctl and 16 SM-Cn −/− Ang-II-treated mice. Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and s.e.m. **** p < 0.0001, *** p < 0.001; repeated-measurements (RM) two-way ANOVA with Tukey’s post hoc test. Red crosses (†) indicate mice that died before completion of the experiment and exhibited a ruptured aneurysm. ( E ) Representative images of aortas from mice treated as indicated. Scale bar, 1 mm. ( F ) Representative images of hematoxylin–eosin (HE), Masson trichrome (Masson), and Van Gieson (VG) staining on AbAo sections from 4 Cn-Ctl, 3 EC-Cn −/− , and 4 SM-Cn −/− mice. Scale bar, 100 µm. Medial area in AbAo sections from ( G ) 7 Cn-Ctl and 7 EC-Cn −/− control-treated mice and 16 Cn-Ctl and 7 EC-Cn −/− Ang-II-treated mice and ( H ) 7 Cn-Ctl and 5 SM-Cn −/− control-treated mice and 9 Cn-Ctl and 9 SM-Cn −/− Ang-II-treated mice. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s., non-significant; two-way ANOVA with Šídák post hoc test. Underlying data can be found in . Data from a subset of untreated and Ang-II-treated Cn-Ctl mice were repeated in panels C and D and in panels G and H as indicated in .

Article Snippet: For the AAA model, Cn-Ctl, SM-Cn −/− , and EC-Cn −/− male mice were fed a HFD (EF M HF coconut oil, + 7.5 g/kg Cholesterol Experimental diet, 38% chow, 10 mm; S9167-E011, SSNIFF) for 12 consecutive weeks.

Techniques: Control, Staining