recombinant human igfbp6  (R&D Systems)

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: Frontiers in Molecular Biosciences

Article Title: Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

doi: 10.3389/fmolb.2022.905468

Figure Lengend Snippet: IGFBP-6 shows higher expression in F508del-CFTR CFBE cells and is further increased after LPS treatment. (A) Total RNA was extracted and qRT-PCR was performed in order to quantify IGFBP-6 mRNA normalized to GADPH as housekeeping gene. Data represent the mean ± SEM ( n = 3). Statistical significance tested using paired two-tailed t -test. (B) Immunoblots of basal expression of IGFBP-6 in Wt and F508del-CFTR CFBE cells. (C) Data represent the mean ± SEM of the ratio IGFBP-6/Calnexin ( n = 3). Statistical significance tested using paired two-tailed t -test. (D,E) Cells were treated with 1 μg/ml LPS for 4 h. Total RNA was extracted and qRT-PCR was performed in order to quantify IGFBP-6 mRNA s normalized first to GADPH as housekeeping gene and then to control untreated cells. Data represent the mean ± SEM ( n = 4). Statistical significance tested using two-way ANOVA with Tukey’s multiple comparisons test (F) Immunoblots of IGFBP-6 following treatment with 1 μg/ml LPS for 24 h. (G) Data represent the mean ± SEM of the ratio IGFBP-6/Calnexin normalized to Calnexin control ( n = 3–4). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Cells were treated for 24 h with 0.1% DMSO or 200 ng/ml IGFBP-6 (PeproTech) or 3 µM VX-661 + 3 µM VX-445 (Selleck Chemicals) +/− 200 ng/ml IGFBP-6.

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

Journal: Frontiers in Molecular Biosciences

Article Title: Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

doi: 10.3389/fmolb.2022.905468

Figure Lengend Snippet: IGFBP-6 is reduced by Dimethyl fumarate. (A) Cells were treated with 0.1% DMSO, 1 μg/ml LPS ± 50 µM DMF or (B) PBS, 30 ng/ml IL-1β + 30 ng/ml TNFα ± 50 µM DMF for 4 h. Total RNA was extracted and qRT-PCR was performed in order to quantify IGFBP mRNA normalized to GADPH as housekeeping gene and then to control untreated cells. Data represent the mean ± SEM ( n = 4). (C) IGFBP-6 secretion after stimulation with 0.1% DMSO, 1 μg/ml LPS, 30 ng/ml IL-1β + 30 ng/ml TNFα ± 50 µM DMF for 24 h. Data represent the mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical significance tested using two-way ANOVA with Tukey’s multiple comparisons test to the control (Wt or F508del) for each cell line.

Article Snippet: Cells were treated for 24 h with 0.1% DMSO or 200 ng/ml IGFBP-6 (PeproTech) or 3 µM VX-661 + 3 µM VX-445 (Selleck Chemicals) +/− 200 ng/ml IGFBP-6.

Techniques: Quantitative RT-PCR

Journal: Frontiers in Molecular Biosciences

Article Title: Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

doi: 10.3389/fmolb.2022.905468

Figure Lengend Snippet: Anti-IGFBP-6 antibodies increased pro-inflammatory cytokines expression. (A) F508del-CFTR CFBE cells were treated with PBS or 0.2–200 ng/ml IGFBP-6 for 4 h. Statistical significance tested using two-way ANOVA with Tukey’s multiple comparisons test. (B) Cells were treated with 1 μg/ml LPS ± an antibody directed against IGFBP-6 (1 μg/ml) for 4 h. Total RNA was extracted and qRT-PCR was performed in order to quantify IL-1β, IL-6 and TNF-α mRNAs normalized to GADPH as housekeeping gene and then to control untreated cells. Data represent the mean ± SEM ( n = 3). Statistical significance tested using paired two-tailed t -test.

Article Snippet: Cells were treated for 24 h with 0.1% DMSO or 200 ng/ml IGFBP-6 (PeproTech) or 3 µM VX-661 + 3 µM VX-445 (Selleck Chemicals) +/− 200 ng/ml IGFBP-6.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Molecular Biosciences

Article Title: Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

doi: 10.3389/fmolb.2022.905468

Figure Lengend Snippet: IGFBP-6 shows higher expression in F508del / F508del nasal epithelial cells from three CF donors and reduces pro-inflammatory cytokines expression. (A) Total RNA was extracted from 2 patients homozygous for F508del mutation and 2 non-CF donors. qRT-PCR was performed in order to quantify IGFBP-6 mRNA normalized to GADPH as housekeeping gene. Data represent the mean ± SEM ( n = 4). Statistical significance tested using paired two-tailed t -test (B) Cells were treated with 0.1% DMSO, 1 μg/ml LPS, 30 ng/ml IL-1β + 30 ng/ml TNFα ± 50 µM DMF for 4 h. Total RNA was extracted and qRT-PCR was performed in order to quantify IGFBP mRNA normalized to control untreated cells. Data represent the mean ± SEM ( n = 4). (C) Cells were treated with 200 ng/ml IGFBP-6 for 4 h. Total RNA was extracted and qRT-PCR was performed in order to quantify IL-1β, IL-6 and TNFα mRNAs normalized to control untreated cells. Data represent the mean ± SEM ( n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical significance tested using two-way ANOVA with Tukey’s multiple comparisons test to the control for (Wt or F508del) each cell line.

Article Snippet: Cells were treated for 24 h with 0.1% DMSO or 200 ng/ml IGFBP-6 (PeproTech) or 3 µM VX-661 + 3 µM VX-445 (Selleck Chemicals) +/− 200 ng/ml IGFBP-6.

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Two Tailed Test