Additional file 1 of Aging aggravates acetaminophen-induced acute liver injury and inflammation through inordinate C/EBPα-BMP9 crosstalk
Creators: name: Liu, Rui
name: Xu, Wentao
name: Zhu, He
name: Dong, Zijian
name: Dong, Huke
name: Yin, Shi
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 1: Table S1. Primer sequences used for real-time RT-PCR. Figure S1. Supplementary figure related to Figure 2. (A-B) Rm-BMP9 was injected into the tail vein, the APAP-ALI model was established 1 h later, and 24 h later, serum and liver tissue were collected from each group of mice. Representative images showing TUNEL staining (A) and the mRNA expression levels of related cytokines (Il1b, Il6 and Tnfa) (B) in each group. (C-D) An APAP-ALI model was established with young and aged WT mice and with young and aged Bmp9-/- mice. Representative images showing TUNEL staining (C) and the mRNA expression levels of related cytokines (Il1b, Il6 and Tnfa) (D) in each group. (E) IF staining for P-SMAD1/5/9 in young and aged WT mice and aged Bmp9-/- mice. The average target gene/Gapdh ratios of different experimental groups relative to the control group. GAPDH was used as the loading control for immunoblotting. *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001. Figure S2. BMP9 expression is regulated by C/EBPα in vivo. (A-C) Cebpa-overexpressing AAV2/8 was injected into the mouse tail vein 2 weeks after the models were established. (A) IF and IHC staining for C/EBPα in liver slices. (B) mRNA expression levels of Cebpa and Bmp9. (C) Protein expression levels of C/EBPα and BMP9. The average target gene/Gapdh ratios of different experimental groups relative to the control group. GAPDH was used as the loading control for immunoblotting. *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001. Figure S3. Supplementary figures related to Figure 4. (A-B) Additional mRNA expression level measurements (Cxcl1, Cxcl13, Mcp1, Arg1 and Il10). (C) Survival curves of mice in the Cebpa-overexpressing and control groups when the dose of APAP treatment was increased to 500 mg/kg. (D) mRNA expression level measurements (Cebpa, Bmp9, Atg3 and Atg7). The average target gene/Gapdh ratios of different experimental groups relative to the control group. *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001. Figure S4. (A) LC3 I/II and BMP9 levels in primary hepatic MΦs from young and aged mice treated with APAP-treated AML-12 cell supernatant (Sup) and/or RAPA and/or BA. GAPDH was used as the loading control for immunoblotting. Figure S5. BMP9 increases etoposide-induced macrophage senescence and proinflammatory cytokine expression. (A) mRNA expression levels of p21, p53, Il1b, Il6 and Tnfa. (B) SA-β-gal staining for iBMDMs after control treatment, etoposide treatment for 1 day, etoposide treatment for 2 days, or etoposide plus Rm-BMP9 treatment for 2 days. (C) mRNA expression levels of iNos, Il1b, Il6 and Tnfa in hepatic MΦs isolated from Bmp9-/- and WT mice. The average target gene/Gapdh ratios of different experimental groups relative to the control group. *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001. Figure S6. (A) Representative images showing IF staining for iNOS (red) and CD206 incorporation (green) with DAPI counterstaining (blue) in each iBMDM group. (B) Representative images showing BMP9 IF staining of primary MΦs isolated from mouse livers treated after AAV-F4/80 or AAV-F4/80-Bmp9 injection.