Journal: PLOS ONE

Article Title: Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway

doi: 10.1371/journal.pone.0304365

Figure Lengend Snippet: Primer pair sequence of target genes.

Article Snippet: PKA antibodies, Col-I antibodies, FN antibodies, and α-SMA antibodies are bought from Proteintech LTD. IL-1β antibody, NLRP3 antibody, and Caspase-1 antibody are ordered from Beijing Bioss Company.

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway

doi: 10.1371/journal.pone.0304365

Figure Lengend Snippet: (A) Effect of AS-IV on α-SMA immunofluorescence expression in renal tubular epithelial cells after UII intervention. (B) Effect of AS-IV on FN immunofluorescence expression in renal tubular epithelial cells after UII intervention. (C) Effect of AS-IV on α-SMA and FN protein expression in renal tubular epithelial cells after UII intervention. (D) Effect of AS-IV on mRNA expression of α-SMA and FN in renal tubular epithelial cells after UII intervention. * means p <0.05 compared with group C, # means p <0.05 compared with group UⅡ. (scale bar: 36.8 μm). The experiment was repeated three times.

Article Snippet: PKA antibodies, Col-I antibodies, FN antibodies, and α-SMA antibodies are bought from Proteintech LTD. IL-1β antibody, NLRP3 antibody, and Caspase-1 antibody are ordered from Beijing Bioss Company.

Techniques: Immunofluorescence, Expressing

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 1: Molecular structures and functions of mouse PGC-1a isoforms. (A) Gene and predicted protein structures of PGC-1a isoforms. (B) C2C12 myotubes infected with adenoviruses encoding individual PGC-1a isoforms or b-galactosidase (b-gal, control) were subjected to RT-qPCR analysis of carnitine palmitoyltransferase 1b (Cpt1b), PPARa (Ppara), mitochondrial ATP synthase subunit b (Atp5b), glucose transporter 4 (Glut4), mitochondrial transcription factor A (Tfam), and cytochrome c oxidase subunit II (Mt-Co2) mRNAs (n ¼ 8 independent experiments). The amount of each mRNA was normalized by that of 36B4 mRNA, and normalized values are expressed relative to the corresponding value for myotubes expressing b-gal. (C) COS7 cells transfected with expression vectors encoding each PGC-1a isoform (or with the corresponding empty vector, Control) and with an expression vector for mouse PPARa were subjected to immunoprecipitation (IP) with antibodies to PGC-1a, and the resulting precipitates were subjected to immunoblot analysis with antibodies to PPARa (upper panel) or to PGC-1a (lower panel). (D) Luciferase reporter assay with the reporter plasmid 3x-PPRE-luc for the transcriptional coactivator activity of PGC-1a isoforms expressed together with PPARa in C2C12 cells (n ¼ 4 independent experiments). (E, F) C2C12 myotubes infected with adenoviruses encoding PGC-1a isoforms or b-gal (control) were subjected to immunoblot analysis of PGC-1a and GAPDH as a loading control (E) as well as assayed both for respiration rate under basal conditions and in the presence of oligomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and for mitochondrial proton leak (F) (n ¼ 8 independent experiments). (G) Effects of clenbuterol on the promoter activity of PGC-1aa or PGC-1ab/c genes in C2C12 myoblasts. All quantitative data are means s.e.m. *P < 0.05 versus the corresponding value for b-gal (B) or for the indicated comparisons (D, F) by one-way (B, F) or two-way (D, G) ANOVA with Bonferroni’s post hoc test. NS, not significant.

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Infection, Control, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 2: Effects of exercise on the abundance of PGC-1a isoform mRNAs in mice and humans. (A) RT-qPCR analysis of PGC-1a isoform mRNAs in skeletal muscle of male C57BL/6J mice both under the static condition (Ex ()) and after exercise on a treadmill at 15 m/min for 120 min (Ex (þ)) (n ¼ 8 mice per group). EDL, extensor digitorum longus. (B) RT-qPCR analysis of PGC-1a isoform mRNAs in vastus lateralis skeletal muscle of human individuals with NGT (n ¼ 10) or T2D (n ¼ 10) both under the static condition (Ex ()) and after ergometer exercise for 30 min (Ex (þ)). PGC-1a isoform mRNAs were subjected to quantification with the use of standard curves determined with plasmids containing the corresponding cDNAs so as to allow comparison of their amounts, and copy number was determined by RT-qPCR. The relative copy number of the transcripts was normalized to that of 36B4. Data in (A) and (B) are means s.e.m. *P < 0.05, **P < 0.01, and NS by one-way ANOVA with Bonferroni’s post hoc test. (C) Correlation of log2[fold change] for the exercise-induced increase in the abundance of PGC-1a isoform mRNAs in human skeletal muscle and VO2 max (NGT, n ¼ 10; T2D, n ¼ 10). Spearman correlation R2 values and P values are indicated.

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Comparison

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 3: Obesity and insulin resistance in PGC-1aAE1KO mice. (AeD) Body mass at the indicated ages (n ¼ 16) (A), tissue mass at 4 months of age (n ¼ 16) (B), as well as abdominal images and adipose tissue mass (n ¼ 4) (C) and lean body mass (n ¼ 4) (D) obtained by CT at 4 months of age for WT and KO mice. (EeG) Hematoxylineeosin staining of eWAT (E) as well as adipocyte diameter in epididymal or subcutaneous fat as determined either with a Coulter counter (F) or by histological analysis (G) for WT and KO mice at 3 months of age (n ¼ 4). (H) Food intake for 4-month-old WT and KO mice (n ¼ 10). (I, J) Violin plots for blood glucose (I) and plasma insulin (J) concentrations in the randomly fed state and at the indicated ages (n ¼ 16). Plot center lines denote the median. (K, L) Blood glucose (K) and plasma insulin (L) levels during an intraperitoneal GTT (left panels) as well as the corresponding area under the curve (AUC) values (right panels) for WT and KO mice at 7 months of age (n ¼ 11). (M, N) RT-qPCR analysis of total PGC-1a mRNA (n ¼ 8) (M) and qPCR analysis of mtDNA content (n ¼ 6) (N) for the indicated skeletal muscles of WT and KO mice at 4 months of age. (O) Function of mitochondria isolated from gastrocnemius muscle of WT and KO mice at 4 months of age (n ¼ 4). Quantitative data are means s.e.m. for the indicated numbers (n) of mice in (AeD, G, HeO). *P < 0.05, **P < 0.01, and NS versus the corresponding WT value or for the indicated comparisons by the two-tailed unpaired Student’s t test (BeD, HeJ; K,L (right panels)) or by one-way (A; K, L (left panels); M-O) or two-way (G) ANOVA with Bonferroni’s post hoc test.

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Staining, Clinical Proteomics, Quantitative RT-PCR, Muscles, Isolation, Two Tailed Test

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 4: Oxygen consumption, heat production, and locomotor activity during the light and dark phases for PGC-1aAE1KO mice. (A) The circadian pattern of locomotor activity for WT and KO mice at 3 months of age was analyzed with an infrared sensor for 10 days (n ¼ 10). Locomotor activity is expressed as a percentage of the daily total: 100% (activity counts for each hour/total activity counts for 24 h). Each point corresponds to an individual mouse. (B) RT-qPCR analysis of PGC-1a isoform mRNAs in skeletal muscle of WT and KO mice during the light and dark phases in the absence or presence of a running wheel (n ¼ 4). (CeE, GeI) Circadian pattern of oxygen consumption (VO2) (C, G) as well as the levels of oxygen consumption (D, H) and heat production (E, I) during the light and dark phases in the absence (WT, n ¼ 7; KO, n ¼ 9) (CeE) or presence (WT, n ¼ 5; KO, n ¼ 7) (GeI) of a running wheel for 4-month-old WT and KO mice. (F, J) Locomotor activity for 4-month-old WT and KO mice during the light and dark phases was analyzed for 4 days in the absence (WT, n ¼ 7; KO, n ¼ 9) (F) or presence (WT, n ¼ 5; KO, n ¼ 7) (J) of a running wheel. The center lines of the violin plots indicate the median. Data are means s.e.m. in (BeE, GeI), and n values indicate the numbers of mice. *P < 0.05, **P < 0.01, and NS by the two-tailed unpaired Student’s t test (DeF, HeJ) or two-way ANOVA with Bonferroni’s post hoc test (B).

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Activity Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 5: Impaired motor performance as well as attenuated exercise-induced energy expenditure and gene expression in PGC-1aAE1KO mice. (AeC) Motor per- formance of 3-month-old WT and KO mice (n ¼ 8) was assessed by an exercise endurance test with stepwise increases in treadmill rate (A). Exhaustion was defined as the inability of the animal to remain on the treadmill despite mechanical prodding. The average time (B) and distance (C) of running until exhaustion were determined. (DeG) Time course of oxygen consumption (n ¼ 8) (D), total oxygen consumption (n ¼ 8) (E), carbohydrate oxidation rate (n ¼ 6) (F), and lipid oxidation rate (n ¼ 6) (G) during the first 30 min of forced treadmill exercise (Ex) at 25 m/min for 4-month-old WT and KO mice. (H, I) Percentage change in body mass (n ¼ 14) after (H), and epididymal fat mass (n ¼ 4) before and after (I), forced treadmill exercise for 120 min at 15 m/min for 4-month-old WT and KO mice. (J) Intramuscular temperature of 4-month-old WT and KO mice during forced treadmill exercise at 15 m/min (n ¼ 5). (K, L) RT-qPCR analysis of PGC-1a isoform mRNAs in gastrocnemius muscle (n ¼ 6) (K) and immunoblot analysis of PGC-1a in EDL (n ¼ 3) (L) for 4-month-old WT and KO mice under the static condition (Ex ()) or after forced treadmill exercise at 15 m/min for 120 min (Ex (þ)). (M) RT-qPCR analysis of mRNAs for the indicated genes in gastrocnemius muscle of 4-month-old WT and KO mice under the static condition or after forced treadmill exercise at 15 m/min for 120 min (n ¼ 4). The amount of each mRNA was normalized by that of 36B4 mRNA, and normalized values are expressed relative to the corresponding value for WT Ex (). All quantitative data are means s.e.m. for the indicated numbers (n) or mice with the exception of those in (A) and (H), with the median being indicated in (H). *P < 0.05, **P < 0.01, and NS by the two-tailed unpaired Student’s t test (B, C, H) or two-way ANOVA with Bonferroni’s post hoc test (EeG, I, K, M).

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 6: Characterization of BAT and change in body temperature in response to cold exposure in PGC-1aAE1KO mice. (A, B) Hematoxylineeosin staining (A) and tissue mass (n ¼ 16) (B) for interscapular BAT of WT and KO mice at 8 weeks of age. (C, D) RT-qPCR analysis of PGC-1a isoform mRNAs (n ¼ 4) (C) and of UCP1 mRNA (n ¼ 4) (D) in BAT of 6-week-old WT and KO mice maintained either at 22 C or for the indicated times (C) or 3 h (D) at 4 C. The amount of UCP1 mRNA was normalized by that of 36B4 mRNA, and normalized values are expressed relative to the value for WT at 22 C. (E) Rectal temperature of 6-week-old WT and KO mice during exposure to 4 C for the indicated times (n ¼ 6). (F, G) RT-qPCR analysis of PGC-1a isoform mRNAs (F) and of expression of the indicated genes (G) in eWAT of 4-month-old WT and KO mice maintained at 22 C or for 3 h at 4 C (n ¼ 4). Gene expression in (G) was normalized by the amount of 36B4 mRNA, and normalized values are expressed relative to the value for WT at 22 C. All quantitative data are means s.e.m. for the indicated numbers (n) or mice. *P < 0.05, **P < 0.01, and NS versus the corresponding value for KO mice (E) or for the indicated comparisons (BeD, F, G) by the two-tailed unpaired Student’s t test (B) or two-way ANOVA with Bonferroni’s post hoc test (CeG).

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Staining, Quantitative RT-PCR, Expressing, Gene Expression, Two Tailed Test

Journal: Molecular metabolism

Article Title: Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

doi: 10.1016/j.molmet.2024.101968

Figure Lengend Snippet: Figure 7: Effects of exercise training on skeletal muscle remodeling in PGC-1aAE1KO mice. (A, B) RT-qPCR analysis of MHC mRNAs (n ¼ 6) (A) and qPCR analysis of mtDNA (n ¼ 8) (B) in gastrocnemius of WT and KO mice maintained with or without exercise training for 6 weeks beginning at 3 months of age. The amount of each MHC mRNA was normalized by that of 36B4 mRNA, and normalized values are expressed relative to the corresponding value for training (). (C, D) Vascular density in EDL as determined by immunohistochemical staining with antibodies to CD31 (C) and represented by the average number of CD31-positive capillaries per high-power field (HPF) among four such HPFs (D) for WT and KO mice (n ¼ 4) maintained with or without exercise training for 6 weeks beginning at 3 months of age. Mice at 4 months of age subjected to forced treadmill exercise at 15 m/min for 120 min were also analyzed for comparison (Ex (þ), n ¼ 4). (E) RT-qPCR analysis of PGC-1a isoform mRNAs in gastrocnemius of WT and KO mice as in (A) (n ¼ 6). All quantitative data are means s.e.m. for the indicated numbers (n) of mice. *P < 0.05, **P < 0.01, and NS by two-way ANOVA with Bonferroni’s post hoc test. (F) Model for the roles of PGC-1a isoforms in skeletal muscle. Circle sizes indicate the relative abundance of PGC-1a isoforms.

Article Snippet: COS7 cells transfected with pcDNA3.1-based expression vectors encoding mouse PGC-1a isoforms and mouse PPARa were subjected to immunoprecipitation with antibodies to PGC-1a (ST1202, Calbiochem), and the resulting precipitates were subjected to immunoblot analysis with the same antibodies to PGC-1a and with those to PPARa (H-08, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Immunohistochemical staining, Staining, Comparison

Journal: International immunopharmacology

Article Title: Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

doi: 10.1016/j.intimp.2024.112176

Figure Lengend Snippet: Fig. 1. Endothelial cells produce alarmin IL-1α to promote lung fibrosis. (A-B) Re-analysis of published scRNA-seq dataset of bleomycin-treated mouse lungs from Gene Expression Omnibus (GEO) (GSE132771). (A) All cell clusters were identified using the T-distributed Stochastic Neighbor Embedding (t-SNE) technique. (B) IL-1α expressing cell populations were exhibited by feature plot. Vascular endothelial cells were identified by representative markers Pecam1 and Ednrb. Macrophages were identified by representative markers Ptprc and Adgre1. The color code in the t-SNE plot of IL-1α+ cell types overlapped with Pecam1+ Ednrb+ cells clusters and Ptprc+ Adgre1+ cell clusters. (C) Strategy for endothelial cell-specific deletion of IL-1α in adult mice. Mice expressing inducible endothelial cell-specific VE-Cadherin-CreERT2 were bred with Il1aLoxP/LoxP mice. Resulting VE-Cadherin- CreERT2Il1aLoxP/LoxP mice were injected with tamoxifen to induce specific ablation of Il1a gene in endothelial cells (Il1aiΔEC/iΔEC). Primary lung vascular endothelial cells were isolated and the knockout efficiency of IL-1α was determined by western blot.(D) Strategy for endothelial cell-specific deletion of IL-1α before bleomycin injection. Il1αloxP/loxPCreERT2 mice were subjected to daily injection of tamoxifen for six consecutive days, then mice were rested for another six days. To induce lung fibrosis, mice were subjected to an intratracheal instillation of bleomycin (2.0 mg/kg) on day 0 and euthanized at day 21. (E) Representative photomicrographs of lung sections stained with Masson’s trichrome and H&E, magnification 20×, scale bars 50 μm.(F) Collagen contents in the right mouse lungs of indicated groups were measured by hydroxyproline assay.(G) α-smooth muscle actin (SMA) and connective tissue growth factor (CTGF) protein levels in the described lung tissue ho mogenates were examined by western blot.(H) Schematic demonstrated IL-1α functions as an alarmin released from injured endothelial cells and promotes the progression of lung fibrosis.

Article Snippet: The following antibodies were used: α-SMA (Proteintech, 14395–1- A, 1:5000), CTGF(Abcam, ab6992, 1:2000), IL-1α (Proteintech, 16765–1-AP, 1:500), CASP8(Proteintech, 66093–1-Ig, 1:500), RIPK1 (HUABIO, ET1701-79, 1:1000), PANX1(Affinity Biosciences, DF8316, 1:1000), Flag tag(CST, 14793, 1:2000), HA tag(Affinity Biosciences, T0050, 1:2000), β-actin(Proteintech, 66009–1-Ig, 1:3000), Anti-rabbit IgG, HRP-linked (Proteintech, SA00001-2, 1:3000), Anti-mouse IgG, HRP-linked (Proteintech, SA00001-1, 1:3000).

Techniques: Gene Expression, Expressing, Injection, Isolation, Knock-Out, Western Blot, Staining, Hydroxyproline Assay

Journal: International immunopharmacology

Article Title: Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

doi: 10.1016/j.intimp.2024.112176

Figure Lengend Snippet: Fig. 2. Endothelial cell-derived IL-1α facilitates the progression of fibrosis at the early stage of lung repair. Data are shown as mean ± SEM (n = 6–––8 mice/group) and representative of three independent experiments (*p < 0.05, ** p < 0.01). (A) Strategy for endothelial cell-specific deletion of IL-1α before bleomycin injection. At 7th day after final tamoxifen treatment, these mice with endothelial cell- specific deletion of IL-1α were treated with intratracheal instillation of bleomycin (2.0 mg/kg). These mice were designated as day 0 after bleomycin injury (Il1αiΔEC/iΔEC-0d) and euthanized 21 days after bleomycin injury for analysis.(B) Representative photomicrographs of lung sections stained with Masson’s trichrome and H&E, magnification 20×, scale bars 50 μm.(C) Collagen contents in the right mouse lungs of indicated groups were measured by hydroxyproline assay.(D) α-smooth muscle actin (SMA) and connective tissue growth factor (CTGF) protein levels in the described lung tissue homogenates were examined by western blot.(E- F) Strategy for endothelial cell-specific deletion of IL-1α 7 days after bleomycin (Il1αiΔEC/iΔEC-7d) or 14 days after bleomycin (Il1αiΔEC/iΔEC-14d). Mice were subjected to intratracheal instillation of bleomycin (2.0 mg/kg) on day 0 and euthanized at day 21 for analysis.(G) Representative photomicrographs stained with Masson’s trichrome and H&E from the lung sections of Il1αiΔEC/iΔEC-7d and Il1αiΔEC/iΔEC-14d mice.(H) Collagen contents in the right lungs of Il1αiΔEC/iΔEC-7d and Il1αiΔEC/iΔEC- 14d mice were measured by hydroxyproline assay.(I) α-SMA and CTGF in lung tissue homogenates of Il1αiΔEC/iΔEC-7d and Il1αiΔEC/iΔEC-14d mice were examined by western blot.(J) The schematic diagram interpreted that IL-1α produced by endothelial cells plays a crucial role in promoting the initiation or early stages of pulmonary fibrosis.

Article Snippet: The following antibodies were used: α-SMA (Proteintech, 14395–1- A, 1:5000), CTGF(Abcam, ab6992, 1:2000), IL-1α (Proteintech, 16765–1-AP, 1:500), CASP8(Proteintech, 66093–1-Ig, 1:500), RIPK1 (HUABIO, ET1701-79, 1:1000), PANX1(Affinity Biosciences, DF8316, 1:1000), Flag tag(CST, 14793, 1:2000), HA tag(Affinity Biosciences, T0050, 1:2000), β-actin(Proteintech, 66009–1-Ig, 1:3000), Anti-rabbit IgG, HRP-linked (Proteintech, SA00001-2, 1:3000), Anti-mouse IgG, HRP-linked (Proteintech, SA00001-1, 1:3000).

Techniques: Derivative Assay, Injection, Staining, Hydroxyproline Assay, Western Blot, Produced

Journal: International immunopharmacology

Article Title: Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

doi: 10.1016/j.intimp.2024.112176

Figure Lengend Snippet: Fig. 3. Endothelial cells supply alarmin IL-1α to enhance liver fibrosis. Data are shown as mean ± SEM (n = 6–8 mice/group) and representative of three inde pendent experiments (*p < 0.05, **p < 0.01). (A-B) Re-analysis of published scRNA-seq dataset of liver non-parenchymal cells from CCl4-treated mice (GSE145086). (A) All cell clusters were identified by tSNE plots. (B) IL-1α expressing cell populations were exhibited by feature plot function; vascular endothelial cells were identified by representative markers Pecam1 and Lyve1; macrophages were identified by representative markers Ptprc and Adgre1. (C) Protocol for CCl4-induced liver fibrosis and the time points when the tissue samples were collected. (D) Representative photomicrographs of mouse liver sections stained with Sirius red and H&E, magnification 20×, scale bars 50 μm. (E) α-SMA and CTGF protein levels in liver tissue homogenates were examined by western blot. (F) Collagen contents in the mouse liver were measured by hydroxy proline assay. (G) Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels of indicated mouse groups were measured. (H) Serum ALT levels were measured at 0h, 24h and 36h after a single injection of CCl4. (I) The temporal expression of IL-1α in lung homogenates was detected by qRT-PCR in response to single CCl4 injection.(J) The expression of Collagen I was detected by immunofluorescence staining, endothelial cell-derived IL-1α knockout alleviates the formation of CollagenI at 24h after a single injection of CCl4.(K) Serum ALT levels indicated that endothelial cell-derived IL-1α KO restores liver function at 24h after a single injection of CCl4.

Article Snippet: The following antibodies were used: α-SMA (Proteintech, 14395–1- A, 1:5000), CTGF(Abcam, ab6992, 1:2000), IL-1α (Proteintech, 16765–1-AP, 1:500), CASP8(Proteintech, 66093–1-Ig, 1:500), RIPK1 (HUABIO, ET1701-79, 1:1000), PANX1(Affinity Biosciences, DF8316, 1:1000), Flag tag(CST, 14793, 1:2000), HA tag(Affinity Biosciences, T0050, 1:2000), β-actin(Proteintech, 66009–1-Ig, 1:3000), Anti-rabbit IgG, HRP-linked (Proteintech, SA00001-2, 1:3000), Anti-mouse IgG, HRP-linked (Proteintech, SA00001-1, 1:3000).

Techniques: Expressing, Staining, Western Blot, Injection, Quantitative RT-PCR, Immunofluorescence, Derivative Assay, Knock-Out