Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

Journal: Biology of reproduction

Article Title: Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

doi: 10.1095/biolreprod.106.058867

Figure Lengend Snippet: FIG. 1. Detection of IGFBP7 in ovarian follicular fluid, cultured GCs and the conditioned medium of GCs, and ovarian sections. A) Detection of IGFBP7 in follicular fluid (FF) and uterine fluid (UF). After collecting the FF and UF from immature rats 2 days after eCG treatment, the samples were concentrated and subjected to SDS-PAGE followed by Western blot analysis using a polyclonal anti-IGFBP7 antibody. The protein content in each sample used for Western blot was equivalent to 0.1% (FF, #1) or 0.25% (FF, #2) of the ovary and 0.3% (UF, #1) of the uterus. #1, #2, independent experiment number. B) Detection of IGFBP7 in GCs and in the conditioned medium of GC cultures. GCs were cultured for 24 h with serum-free DMEM and then lysed and immunoprecipitated with anti- IGFBP7 antibody (left panel) in two independent experiments (#1 and #2). The conditioned medium sample (IP[]) was incubated with normal rabbit IgG instead of anti-IGFBP7 antibody. All samples were subjected to Western blotting analysis with anti-IGFBP7 antibody. C) Localization of Igfbp7 mRNA in the ovaries of immature rats 2 days (left panel) or 5 days (right panel) after eCG treatment. Paraffin-embedded sections of ovaries were hybridized with an antisense cRNA probe. Signals were detected in growing follicles (left panel) and in the corpus luteum (right panel). TI, Theca interna; CL, corpus luteum; Bars ¼ 100 lm.

Article Snippet: Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively.

Techniques: Cell Culture, SDS Page, Western Blot, Immunoprecipitation, Incubation

Journal: Biology of reproduction

Article Title: Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

doi: 10.1095/biolreprod.106.058867

Figure Lengend Snippet: FIG. 2. Effect of IGFBP7 treatment on FSH-stimulated 17b-E2 production in rat GCs. Subconfluent GCs were cultured for 24 h in DMEM supplemented with FSH (25 ng/ml) and androstendione (30 ng/ml) in the presence of various doses of IGFBP7 (0–30 ng/ml). 17b-Estradiol levels in the medium were determined 24 h later. The results are presented as means 6 SEM of four independent experiments. #P , 0.001 vs. no treatment; *P , 0.01 vs. FSH alone.

Article Snippet: Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively.

Techniques: Cell Culture

Journal: Biology of reproduction

Article Title: Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

doi: 10.1095/biolreprod.106.058867

Figure Lengend Snippet: FIG. 3. Effect of IGFBP7 treatment on FSH-stimulated Cyp19a1 mRNA expression in rat GCs. Subconfluent GCs were cultured for 24 h with DMEM supplemented with FSH (25 ng/ml) and androstendione (30 ng/ml) in the presence of various doses of IGFBP7 (0–30 ng/ml). Cyp19a1 mRNA levels were determined by semiquantitative RT-PCR analysis using the poly(A)þ

Article Snippet: Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

doi: 10.1095/biolreprod.106.058867

Figure Lengend Snippet: FIG. 4. Effect of IGFBP7 on FSH- and/or activin-stimulated steroidogenesis and ste- roidogenic enzyme expression in GCs. Subconfluent GCs were cultured for 24 h with DMEM supplemented with 30 ng/ml androstendione together with 25 ng/ml FSH and/or 25 ng/ml activin A in the presence of various doses of IGFBP7 (0–30 ng/ml). A) 17b-Estradiol and progesterone levels. The results of three independent experi- ments are expressed as means 6 SEM. #P , 0.001 vs. FSH alone; *P , 0.01 vs. FSH/activin A. B) Cyp19a1 and Cyp11a1 mRNA levels. Semiquantitative RT-PCR analysis was performed using poly (A)þ

Article Snippet: Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

doi: 10.1095/biolreprod.106.058867

Figure Lengend Snippet: FIG. 5. Effect of IGFBP7 knockdown on FSH-stimulated Cyp19a1 and Cyp11a1 mRNA expression and E2 production. The cells were transfected with Igfbp7 siRNA (10 pmol) for 24 h and then cultured for 24 h in the absence () or presence (þ) of FSH. A) Representative RT-PCR analyses. Poly (A)þ RNA was subjected to semiquantitative RT-PCR analysis for Igfbp7, Cyp19a1, Cyp11a1, and G3pdh expression. G3PDH served as an internal control. B) Densito- metric analyses of the Cyp19a1 and Igfbp7 mRNA levels in each treatment. These values from four independent experiments were normalized relative to G3pdh mRNA levels and expressed as a ratio relative to the density of the band in FSH () in the control siRNA-treated group. *P , 0.05 vs. FSH () in control siRNA; #P , 0.05 vs. FSH (þ) in control siRNA or FSH () in Igfbp7 siRNA (C) 17b-E2 levels in the culture medium. The data are expressed as ratios relative to the density of the no-treatment group. Each value is the mean 6 SEM from four independent experiments. *P , 0.05 vs. FSH () in control siRNA.

Article Snippet: Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively.

Techniques: Knockdown, Expressing, Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control

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