Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Volcano plot showing the distribution of DEGs. Yellow dots indicate significantly up-regulated genes, green dots indicate significantly down-regulated genes, and pink dots indicate non-differentially expressed genes. b Heat map of relevant DEGs between fibrosis and control groups. c Top 20 most significantly differentially expressed genes, with IGFBP6 showing the highest significance. d Validation using dataset GSE130123 . CCL4-induced 4 weeks hepatic fibrosis model in mice. (control group n = 4, and fibrosis group n = 5). e IHC staining of COL1A1 and IGFBP6 in hepatic sections from control mice and mice at 4 or 8 weeks after intraperitoneal TAA injection (scale bar: 25 μm). Schematic diagram generated by Biorender (agreement number: AR28HM9B0Q). f Primary HEPs ( n = 6) and HSCs ( n = 6) were isolated from the same liver. The mRNA expression of mouse Igfbp6 . g Primary HSCs were isolated from WT mice treated with three doses of vehicle (PBS, n = 6) or TAA (100 mg/kg, n = 6) every 3 days. Igfbp6 mRNA was measured. h scRNA-seq dataset ( GSE221481 ) from mice with uninjured liver or TAA-induced hepatic fibrosis was analyzed for the expression of Igfbp6 . The fraction of cells expressing Igfbp6 in different cell lineages in liver. Data are expressed as mean ± SD, unpaired Student’s t test, two-tailed.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Control, Biomarker Discovery, Immunohistochemistry, Injection, Generated, Isolation, Expressing, Two Tailed Test

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a qRT-PCR analysis of Acta2 , Fn1 , Col1a1 , and Igfbp6 mRNA levels and b Western blotting analysis of FN, COL1A1, α-SMA, and IGFBP6 protein expression in hepatic tissue homogenates from control and TAA-treated mice at 4 and 8 weeks ( n = 6), Data are expressed as means ± SD, One-way ANOVA. c qRT-PCR analysis of Acta2 , Fn1 , Col1a1 and Igfbp6 mRNA levels in primary mouse HSCs stimulated with TGF-β (10 ng/mL) for 12 h and 24 h or unstimulated ( n = 3), Data are expressed as means ± SD, One-way ANOVA. d Western blotting analysis of protein expression levels of FN, COL1A1, α-SMA and IGFBP6 in primary mouse HSCs stimulated with TGF-β for 24 and 48 h or unstimulated. e –g Western blotting confirmed IGFBP6-knockout in LX2 cells line ( e ). Fibrosis-related gene and protein expression in LX2 cells line stimulated with TGF-β (10 ng/mL) with or without IGFBP6-knockout detected by ( f ) qRT-PCR and ( g ) Western blotting ( n = 3). Data are expressed as means ± SD, Tow-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Knock-Out

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a SwissADME analysis of 30 FDA-approved compounds related to IGFBP6 showing: Heatmap of five drug-likeness rules (Lipinski, Ghose, Veber, Egan, Muegge) and eight pharmacokinetic parameters (GI absorption, BBB permeation, P-gp substrate, CYP1A2/2C19/2C9/2D6/3A4 inhibition). b Bioavailability scores of the compounds. Effect of five candidate compounds on fibrosis-related indicators in TGF-β (10 ng/mL) stimulated primary mouse HSCs were detected ( c ) at 24 h for mRNA levels and ( d ) at 48 h for protein levels ( n = 3). e Chemical structural of PA. f The effect of PA on cell viability of LX2 cells viability was measured using the CCK8 assay ( n = 5). The effect of concentration gradients (0 μM, 0.1 μM, 1 μM, and10μM) of PA against FN, COL1A1, ACTA2, and IGFBP6 in TGF-β (10 ng/mL) -stimulated LX2 cells was examined using ( g ) qRT-PCR for mRNA levels and ( h ) Western blotting for protein levels ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Inhibition, CCK-8 Assay, Concentration Assay, Quantitative RT-PCR, Western Blot

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Three-dimensional binding structure of PA (green) with IGFBP6 (gray ribbon). The Pi-H interactions are depicted as magenta dashed lines. b MST analysis of His-tagged IGFBP6 binding to PA ( n = 3). c CETSA melting curves and derived thermal stability profiles ( n = 3). d Western blotting detection of protein expression levels of FN, COL1A1 and α-SMA in IGFBP6-knockout LX2 cells by PA (10 μM) intervention. The effect of PA’s inhibitory action on TGF-β-induced IGFBP6 overexpression in LX2 cells was assessed by Western blotting in the presence or absence of ( e ) the proteasome inhibitor MG132 or ( f ) the autophagy inhibitor 3-MA ( n = 3). Data are expressed as means ± SD, Two-way ANOVA. g LX2 cells were treated with 1 µM PA in the presence or absence of MG132 for 12 h. IP detection was performed with anti-IGFBP6 antibody, and expression of ubiquitin-coupled IGFBP6 was detected with anti-UB antibody ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Binding Assay, Derivative Assay, Western Blot, Expressing, Knock-Out, Over Expression, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Enrichment analysis results after collection of PA targets via Pharmmapper database and GeneCards database. b Western blotting assay for the influence of PA (10 μM) on different times of TGF-β (10 ng/mL) stimulation-initiated SMAD2/3 phosphorylation levels in primary mouse HSCs ( n = 3). c Effects of different concentrations of PA (0 μM, 0.1 μM, 1 μM, and10 μM) on the phosphorylation level of SMAD2/3 induced by TGF-β (10 ng/mL) action in primary HSCs of mice analyzed by Western blotting ( n = 3). Effect of PA on phosphorylation levels of SMAD2/3 and IGFBP6 in TAA-induced fibrotic hepatic mice in vivo by ( d ) Western blotting and ( e ) IHC ( n = 6, scale bar: 50 μm).

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Western Blot, Phospho-proteomics, In Vivo

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: Phospho-SMAD2/3 levels detected by Western blotting in ( a ) control group and ( b ) IGFBP6-knockout group treated with TGF-β (10 ng/mL) for 15 min, 30 min, or 60 min with or without PA (10 µM) ( n = 3). Data are expressed as means ± SD, One-way ANOVA. c Immunofluorescence analysis of TGF-β (10 ng/mL) -induced SMAD2/3 nuclear translocation in LX2 cells with or without IGFBP6 knockout, treated with or without PA (10 µM) (scale bar: 25 μm). d The mRNA expression levels of Acta2 , Fn1 , and Col1a1 in primary mouse HSCs in the presence or absence of PA or the SMAD3 inhibitor SIS3 were detected by qRT-PCR ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Western Blot, Control, Knock-Out, Immunofluorescence, Translocation Assay, Expressing, Quantitative RT-PCR

Journal: Atherosclerosis Plus

Article Title: The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques

doi: 10.1016/j.athplu.2024.09.001

Figure Lengend Snippet: Expression pattern of components of the STC2 - PAPP-A - IGFBP4 - IGF1 axis. a. Carotid plaques stained by masson trichrome and immunohistochemistry for STC2, PAPP-A, IGFBP4, and IGF1R. Arrowheads point to examples of stained cells. b. Single-cell RNA sequencing data from Wirka et al., 2019, displayed as UMAPs showing annotated cell populations, and expression pattern of PAPP-A , STC2 , IGFBP4 , IGF1R , and IGF1 . SMC = smooth muscle cell; mSMC = modulated SMC; EC = endothelial cell.

Article Snippet: Recombinant human IGFBP4 from RnD Systems (Cat# 804-GB-025) and IGF1 from Austral Biologicals (Cat# GF-050-8) were pre-incubated in media/PBS? and added to all conditioned media from different plaque?-incubations (200 μL) in a final concentration of 1200 ng/mL (of both IGF1 and IGFBP4).

Techniques: Expressing, Staining, Immunohistochemistry, RNA Sequencing Assay

Journal: Atherosclerosis Plus

Article Title: The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques

doi: 10.1016/j.athplu.2024.09.001

Figure Lengend Snippet: Proteolytically active PAPP-A is present in atherosclerotic plaques. a. Study design: Plaque samples from carotid and femoral arteries were incubated in culture media for 24 h, and conditioned media was harvested for analyses. b-c. PAPP-A (b) and STC2 (c) concentration in conditioned media after 24 h of incubation quantified by ELISA. d. Principle of PAPP-A activity assay: Recombinant IGFBP4:IGF1 complex (32 kDa) is incubated with sample containing active PAPP-A resulting in proteolytic cleavage to 14 kDa IGFBP4 fragments. e. Conditioned media from each of the 20 plaque samples were incubated with recombinant IGFBP4:IGF1 for 0 or 24 h and analysed for IGFBP4 cleavage by Western blotting. In all samples, no IGFBP4 fragments are detected at the 0-h time point, but IGFBP4 fragments emerges after 24 h. No PAPP-A activity was detected in the media without tissue incubation (media 24 h). f. Quantitation of PAPP-A activity based on Western blotting. g. Correlation between PAPP-A concentration and -activity. h. Correlation between PAPP-A:STC2 molar ratio and PAPP-A activity. i. PAPP-A:STC2 molar ratio in conditioned media.

Article Snippet: Recombinant human IGFBP4 from RnD Systems (Cat# 804-GB-025) and IGF1 from Austral Biologicals (Cat# GF-050-8) were pre-incubated in media/PBS? and added to all conditioned media from different plaque?-incubations (200 μL) in a final concentration of 1200 ng/mL (of both IGF1 and IGFBP4).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Recombinant, Western Blot, Quantitation Assay