Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 2 | High-fat feeding increases Igfbp7 expression in LMs. a, Diagram showing genes with significant differences in expression in mice with HFD versus ND feeding; n = 3 mice per condition. P value was calculated by Wald test with DESeq2. To set the significance threshold, the adjusted P value was calculated by the Benjamini–Hochberg method with FDR < 0.05. b, Comparison of Igfbp7 expression (normalized RPKM) in mouse liver and LMs; n = 3 mice per condition. c, IGFBP7 protein expression in mouse LMs; n = 3 pooled mice/condition. d, IGFBP7 secretion from mouse LMs assessed by ELISA; n = 5 mice per condition. e, Igfbp7 expression in different cell types in mice; the number of mice (n) is indicated. Hep, hepatocytes; ATMs, adipose tissue macrophages; PM, peripheral macrophages. f, RNA ISH combined with immunohistochemistry (IHC) for mouse liver (scale bars, 20 μm); n = 3 independent experiments with similar results. EC, endothelial cell; H, hepatocyte. g, IGFBP7 RNA editing in human LMs. AA, amino acid. h, Representation of the positions at which RNA editing occurs with respect to the IGFBP7 protein domains. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test.

Article Snippet: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

Techniques: Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Two Tailed Test

Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 4 | Silencing of Igfbp7 in LMs decreases hyperglycaemia and hepatic steatosis. a–d, Glucose tolerance tests (a), overnight fasting glycaemia (b), insulin tolerance tests (c) and liver TG levels (d) in mice treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. e, Representative images of Oil Red O staining performed in mice treated with GeRP-Scr (n = 4) or GeRP-Igfbp7 (n = 5) (scale bars, 100 μm); five random images were taken per mouse with similar results. f,g, Liver transmission electron microscopy (TEM) images (f) and lipid droplet size quantification (g). h, LM and hepatocyte TEM (blue arrowheads indicate lipid droplets); for all TEM experiments, 15 images were randomly acquired from 3 different mice treated with GeRP-Scr or GeRP-Igfbp7. i, NPC LipidTOX staining measured by flow cytometry; n = 5 mice per condition. MFI, median fluorescence intensity. j, Gene set enrichment analysis depicting up- and downregulated pathways in hepatocytes from mice treated with GeRP-Scr (n = 3) or GeRP-Igfbp7 (n = 4) according to MES. The x axis indicates the rank of the pathways according to MES. P value was calculated by permutation test. To set the significance threshold, adjusted P value was calculated by the Benjamini–Hochberg method with FDR < 0.1. k,l, Gene expression pattern of selected genes (normalized RPKM) (k) and gene expression by qPCR (l) in isolated hepatocytes from mice treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. DNL, de novo lipogenesis; HGP, hepatic glucose production. m, Images of ImpL2-Gal4>UAS-mCherry flies showing ImpL2+ cells (red), pHrodo Green staining (green) and overlay (merge). Images to the right represent flies fed a ND or HFD. The green channel was used to visualize structure and for orientation; n = 8 independent experiments with similar results. n,o, ImpL2+ haemocytes counted in 15 randomly selected flies per condition (n) and ImpL2 expression in n = 6 flies per condition (o). p,q, Glucose (p) and TG (q) levels in n = 9 flies per condition. r, Immune gene expression in haemocytes; n = 6 flies per condition. s,t, Glucose (s) and TG (t) levels in ImpL2-RNAi (Hml-Gal4>ImpL2-RNAi) versus W (Hml-Gal4 × w) Drosophila fed a HFD; the number of flies (n) is indicated. A.u., arbitrary units. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test in all panels except o, where one-way ANOVA was used with adjustment for multiple comparisons. DNL, de novo lipogenesis; LD, lipid droplets; HGP, hepatic glucose production.

Article Snippet: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

Techniques: Staining, Transmission Assay, Electron Microscopy, Flow Cytometry, Fluorescence, Gene Expression, Isolation, Expressing, Two Tailed Test

Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 5 | LM-derived IGFBP7 increases lipogenesis and gluconeogenesis. a, Western blots of AKT phosphorylation in primary mouse hepatocytes treated with recombinant IGFBP7 (rIGFBP7) and short-term insulin (Ins); n = 4 biological replicates. p473AKT, AKT phosphorylated at residue 473. b, Blood glucose levels for fasted and refed states in mice fed a ND and treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. c, Western blots of immunoprecipitations in primary mouse hepatocytes for IR, IRS1 and IRS2; n = 3 biological replicates. p-Tyr, phosphorylated tyrosine. d, Western blots and quantification (for a and d) of AKT phosphorylation in primary mouse hepatocytes treated with IR antagonist (IRa) s961, recombinant IGFBP7 and short-term insulin; n = 4 biological replicates. e, Western blots and quantification of AKT phosphorylation in primary mouse hepatocytes treated with recombinant IGFBP7 under hyperinsulinaemia; n = 3 biological replicates. f, Western blots and quantification showing immunoprecipitation of IGFBP7 with IR under hyperinsulinaemia; n = 3 biological replicates. g,h, Western blots and quantification of phosphorylated ERK1/2 (pERK1/2) (g) and immunoprecipitation of Shc with IR (h) in primary mouse hepatocytes treated with recombinant IGFBP7 under hyperinsulinaemia; n = 3 biological replicates. i, Gene expression in primary mouse hepatocytes treated with insulin, recombinant IGFBP7 and the ERK pathway inhibitor U0126 (ERKi); n = 10 technical replicates. j, Image of a human liver spheroid (scale bar, 200 μm). k, Western blots and quantification of AKT and ERK1/2 phosphorylation in human liver spheroids treated with short-term (7-min) or long-term (4-h) insulin and with wild-type (wt) or edited (ed) recombinant IGFBP7 for 4 h. Quantification is expressed as fold change in comparison to control. l, Western blots and quantification of coimmunoprecipitation of IGFBP7 with IR in human liver spheroids under hyperinsulinaemia. Quantification is expressed as fold change in comparison to control. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test in i and by one-way ANOVA with adjustment for multiple comparisons in b and d–h.

Article Snippet: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

Techniques: Derivative Assay, Western Blot, Phospho-proteomics, Recombinant, Residue, Immunoprecipitation, Gene Expression, Comparison, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Release of IGFBP5 in SASP and its effect on senescence. A Representative micrographs of MSCs stained to identify nuclei (DAPI), Ki67 (red), and to evaluate β-galactosidase activity (dark gray). The white arrows indicate senescent cells, which are β-galactosidase positive (β-gal +) and Ki67 negative (Ki67-). We employed a Leica CTR500 microscope, which was equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was captured as a gray-stain using this configuration. This experimental method allowed us to identify cells that exhibited a visible light signal β-galactosidase along with others expressing fluorescent signals within the same cell. CT: untreated cells; CM-IR: cells treated with conditioned medium (CM) collected from irradiated (IR) cells; CM-IR + ab αIGFBP5: cells treated with CM in the presence of IGFBP5 neutralizing antibodies; rIGFBP5: cells incubated with recombinant IGFBP5. The scale bar corresponds to 100 microns. The graph shows the percentage of senescent cells under different experimental conditions. The symbols *** p < 0.001 and * p < 0.05 indicate statistical significance between the control (CT) and treated samples. The symbol ## p < 0.01 indicates statistical significance between the CM-IR sample, chosen as a reference, and CM-IR + ab αIGFBP5. B The graph shows the level of IGFBP5 in CM of MSCs 24 h following irradiation (IR). IR: irradiated MSCs; Anti-oxi: the irradiated MSCs were treated with an anti-oxidant mixture; PXB: irradiated cells were incubated with a drug inhibiting COX2 activity. The symbol ** p < 0.01 indicates statistical significance between the control (CT) and irradiated samples. The symbols ## p < 0.01 and # p < 0.05 indicate statistical significance between the IR sample (second column), chosen as a reference, and IR + Anti-oxi or IR + PXB. The western blot under the graph shows a representative image of IGFBP5 immunodetection. C The graph shows the percentage of senescent cells (β-galactosidase positive and Ki67 negative) under different experimental conditions. rIGFBP5: cells incubated with recombinant IGFBP5; IGFII: cell treated with IGFII either in presence or absence of antibody against IGFIIR (abα IGFIIR); siLRP1: siRNA targeting LRP1 mRNA; siCAV1: siRNA targeting CAVEOLIN-1; siCTR: control siRNA. The symbol ** p < 0.01 indicates statistical significance between the control (first column) and samples (from second to fourth columns) treated with rIGFBP5 and/or IGFII. The symbol °°° p < 0.001 indicates statistical significance between cells treated with abα IGFIIR alone (chosen as reference, see fifth column) and the other samples treated with the antibody in presence of rIGFBP5 and/or IGFII (from sixth to eight columns). The symbols §§§ p < 0.001 indicate and § p < 0.05 indicate statistical significance between the sample treated with siCT (chosen as the reference, see ninth column) and the other siRNA treated samples, reported in columns from the 10th to 12th. The symbol ## p < 0.01 indicates the statistical difference between the sample treated with genistein (Gen) in presence of IGFBP5 and the one treated with IGFBP5 only (second column from left), chosen as reference

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Staining, Activity Assay, Microscopy, Expressing, Irradiation, Incubation, Recombinant, Control, Western Blot, Immunodetection

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: IGFBP5 internalization. A Representative images of MSCs incubated with His-tag-IGFBP5 (IGFBP5-HIS) and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (red). Additionally, CAVEOLIN-1 (CAV1), LRP1, or ITGA2 were stained green. The pictures were taken at 1, 3, and 5 min after IGFBP5 incubation. The inset shows magnified images of IGFBP5 and CAVEOLIN-1 co-localization. The scale bar corresponds to 100 microns. B Representative images of MSCs incubated with His-tag-IGFBP5 and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (green). The pictures were taken at 0, 0.5, 1, 3, 10, 30 and 60 min after IGFBP5 incubation. The scale bar corresponds to 100 microns. C Representative images of Duolink assay to identify physical proximity between IGFBP5 and CAVEOLIN-1. The red staining indicates a close interaction between IGFBP5 and CAVEOLIN-1 5 min following MSCs incubation with His-tag-IGFBP5. The nuclei were stained with DAPI (blue). The scale bar corresponds to 100 microns. D Representative images of MSCs incubated with His-tag-IGFBP5, either in presence or absence of Brefeldin A, and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (red). Additionally, GOLGB1 was stained green. The pictures were taken at 3 min after IGFBP5 incubation. The scale bar corresponds to 100 microns

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Incubation, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Western blot analysis of IGFBP5. Experiments were carried out by cell fractionation into cytoplasmic (cyto) and nuclear fractions. The image shows IGFBP5 levels at different time points following the incubation of MSCs culture with His-tagged IGFBP5. Three different primary antibodies against IGFBP5 were used (see main text). GAPDH and Histone H4 were used as cytoplasmic and nuclear markers, respectively

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Western Blot, Cell Fractionation, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Paracrine action of IGFBP5. A MSCs transfected with control siRNA or IGFBP5-siRNA were X-ray irradiated, and senescence was evaluated 48 h later. The graph depicts the percentage of senescent cells under different experimental conditions. The symbols *** p < 0.001 and * p < 0.05 indicate statistical significance between the control (siCT) and other samples. The # ( p < 0.05) indicateds statistical significance between irradiated siCT versus irradiated siIGFBP5 ( B ) Healthy MSCs were incubated for 48 h with conditioned media (CM) obtained from the previously mentioned samples. The graph illustrates the percentage of senescent cells under different experimental conditions. The symbol *** p < 0.001 indicates statistical significance between the control (siCT) and other samples. The ## ( p < 0.01) indicateds statistical significance between irradiated siCT versus irradiated siIGFBP5. C Representative images of cells stained with anti-γH2AX (green) and His-tag IGFBP5 (red) are shown. Cell nuclei were stained with DAPI. The β-galactosidase activity was evidenced as dark gray. We employed a Leica CTR500 microscope, which was equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was captured as a gray-stain using this configuration. The arrows show cells that were β-galactosidase/γH2AX positive and IGFBP5 negative

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Transfection, Control, Irradiation, Incubation, Staining, Activity Assay, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Interaction between IGFBP5 and retinoic acid receptors. A Cell lysates were immunoprecipitated with either anti-RXRα or anti-RARα antibodies and then subjected to western blot analysis using anti-IGFBP5 antibody. Reciprocal immunoprecipitation (IP) was performed with anti-IGFBP5 antibody, followed by western blots (WB) using either anti-RXRα or anti-RARα antibodies. P and Sup refer to the pellet and supernatant, respectively, of the immunoprecipitation reaction. B Representative images of the Duolink assay to identify the physical proximity between IGFBP5 and RARα. The red staining indicates a close interaction between IGFBP5 and RARα ten minutes after MSCs were incubated with His-tagged IGFBP5. The nuclei were stained with DAPI (blue). The scale bar corresponds to 100 microns. C Recombinant RARα, immobilized on protein A beads, was incubated with IGFBP5 in the presence or absence of ATRA, followed by western blot analysis using anti-IGFBP5 antibody. P and Sup refer to the pellet and supernatant, respectively, of the reaction. D The graph shows the percentage of senescent cells following incubation with IGFBP5 under different experimental conditions. The symbol *** p < 0.001 indicates statistical significance between untreated cells and samples incubated with IGFBP5 (first and second column). The symbols ### p < 0.001 and ## p < 0.01 indicate the statistically significance between the sample treated with IGFBP5 (second column from left), which was chosen as reference, and the others with IGFBP5 in combination with further treatments. E Fluorescence quenching assay. The graphs show the ultraviolet peak emission of IGFBP5 and RARα, due to tryptophan, phenylalanine, and tyrosine, either in the absence or presence of increasing amounts of ATRA, which acted as the quencher. Data are reported with standard deviation. The symbols * p < 0.05, ** p < 0.01 indicate statistical significance between samples incubated with and without ATRA. The latter condition was chosen as the reference

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant, Fluorescence, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Serum IGFBP5 in patients undergoing medical irradiation. The picture illustrates the ELISA analysis of IGFBP5 in the sera of 10 patients before and 48 h after an abdominal CT scan. Each patient is identified by a number. The data are expressed as arbitrary units (A.U.). For every patient, the significant difference between samples harvested before and after CT is denoted with ** ( p < 0.01) or * ( p < 0.05)

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Computed Tomography

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: IGFBP5 signaling in senescence. Healthy cells may become senescent cells following genotoxic stress and then release SASP, which contains IGFBP5. The paracrine action of IGFBP5 may induce secondary senescence in healthy cells not directly affected by genotoxic injury. IGFBP5 can enter cell nuclei through caveolae-dependent endocytosis. Within nuclei, IGFBP5 can interact with RAR/RXR heterodimers and contribute to the transcriptional regulation of genes involved in the executive senescence program

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: