Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 1. IGFBP3 expression inhibited tumor growth and created a hypoxic environment, which induced HIF-2α to overcome the oxygen stress. (A) Heterotransplantation of P4-pBIG2i and P4-pBIG2i-hIGFBP3 in SCID mice. Doxycycline was started to stimulate IGFBP3 at 8 d. Xenograft tumor sizes were measured, and the mice were sacrificed at 12, 15, 22, 28, and 36 d after the implantation. (Five mice per group in each time point. The error bars represent the SDs; ***P < 0.0005.) (B) Photographs of xenograft tumors. (C) Western blot analysis and signal quantitative detections of IGFBP3, HIF-1α, and HIF-2α expressions in xenograft tumors at 15 and 28 d. Cyclophilin A (CypA) as the loading control. The signals of Western blot were quantified and analyzed by Image Studio Lite version 5.2. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 2. Immunohistochemistry staining of xenografts on 12, 15, and 28 d after the tumor implantation. (A) Heterotransplantation tumors P4-pBIG2i and P4-pBIG2i-hIGFBP3 at 12, 15, and 28 d post-tumor implantation on IGFBP3 (100 ×), HIF-1α (40 × and 100 ×), HIF-2α (40 × and 100 ×), HO-1 (100 ×), and VHL (100 ×). P4-pBIG2i xenograft showed no IGFBP3 expressions, while P4-pBIG2i-hIGFBP3 showed a strong IGFBP3 expression after adding doxycycline (started on d 8). HIF-2α increased over time in P4-pBIG2i xenograft, but it only accumulated after 28 d in P4-pBIG2i-hIGFBP3 xenograft. Xenograft tumors without IGFBP3 expressed HIF-2α majorly, especially under a prolonged tumor growth in hypoxic environments. (B) Quantitative analysis of immunohistochemical staining. Images of IHC were taken by Motic Image Plus 3.0 software. The signals of IHC were quantified and analyzed by ImageJ 1.53 k software. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Tumor Implantation, Expressing, Immunohistochemical staining, Software

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 3. Immunocytochemistry staining of A549, P0, and P4 cell cultures under normoxic and hypoxic conditions for 17 h. (A) Under normoxic conditions, P0 expressed more IGFBP3 than P4. P0 showed an increase in HIF-1α, while P4 showed a higher increase in HIF-2α under hypoxia than that under normoxia, indicating that tumor cells without IGFBP3 induced more HIF-2α under acute hypoxic conditions. The A549 cell culture that expressed HIF-2α under hypoxic conditions was used as the study control. (B) Quantitative analysis immunohistochemical staining. Images of ICC were taken by Motic Image Plus 3.0 software. The signals of IHC were quantified and analyzed by ImageJ software. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Immunohistochemical staining, Software

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 4. Protein expression analysis of cell cultures under normoxic and hypoxic conditions. (A) Western blot analysis of IGFBP3, HIF-1α and HIF-2α, HO-1, VHL, and GAPDH expressions in P0, P4, P4 transfectants, and A549 under normoxic and hypoxic conditions. HSP90 was added as the control. (B) Western blot results were transformed into bar figures using Image Studio Lite version 5.2. Signal strengths were quantified and normalized with HSP90. P0 had a higher IGFBP3 expression than P4. In P0 and P4-I, IGFBP3 expression was increased under hypoxia. Accumulation of HIF-1α or HIF-2α correlated with IGFBP3 expressions: a higher IGFBP3 was with a stronger HIF-1α. Both HO-1 and VHL expressions were increased under hypoxia. Each sample was assayed in triplicates, and the experiment was repeated three times independently. (The error bars represent the SDs; **P < 0.001, ***P < 0.0005). Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control, Transformation Assay

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 5. IGFBP3 differentially regulated HIF-1α and HIF-2α synthesis in low and high invasion ability cells. Gene expression and regulation of IGFBP3, HIF-1α, and HIF-2α in cells cultured under normoxic and hypoxic conditions. (A) qPCR analyzed of IGFBP3, HIF-1α, and HIF-2α mRNA expressions in P0 and P4 cultured under normoxic and hypoxic conditions. GAPDH as the normalized control. P4 expressed higher HIF-1α and HIF-2α under normoxia, and P0 expressed lower HIF-2α compare with P4. (B) Luciferase promoter assays on the regulations of IGFBP3, HIF-1α, and HIF-2α promoters in P0 and P4 under normoxic and hypoxic conditions. The table below indicates the presence of endogenous or exogenous IGFBP3. In high-IGFBP3 expression P0, HIF-1α was more activated than HIF-2α at hypoxia. Both HIF-1α and HIF-2α were reduced after depletion of extracellular IGFBP3 by neutralizing antibodies (lanes 4 and 6). In low-IGFBP3 expression P4, both HIF-1α and HIF-2α activities increased under hypoxia. There were no changes in HIF-1α and HIF-2α expressions after adding recombinant hIGFBP3 (lanes 8 and 10). Each sample was assayed and analyzed repeated three times independently. (The error bars represent the SDs; **P < 0.001, ***P < 0.0005.). Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Gene Expression, Cell Culture, Control, Luciferase, Expressing, Recombinant

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 7. Clinical roles of HIF-1α and HIF-2α related to IGFBP3 expression in EOC. In an early stage of EOC, IGFBP3 expresses and inhibits proliferation and angiogenesis. The lack of blood vessels causes tumors to enter a hypoxic state, which stimulates cells to activate HIFs. HIF-1α is the major HIFs in this tumor stage. HIF-1α stimulates angiogenesis, proliferation, and induces IGFBP3 synthesis. The continuous expression of IGFBP3 prolongs the hypoxic state of the tumors by inhibiting tumor vasculogenesis. Prolonged hypoxia causes the promoter to be methylated and silence the expression of IGFBP3. As IGFBP3 decreases, the cells begin to proliferate and switch to activate HIF-2α majorly under hypoxic conditions. HIF-2α plays a significant role in cancer aggressiveness by regulating angiogenesis, invasion, and metastasis. Henceforth, the cancer cells mainly express HIF-2α under hypoxic stress. The decrease in IGFBP3 and the activation of HIF-2α instead of HIF-1α accelerate the progression in EOC. PowerPoint 2016 and Photoshop CS2 version 9 were used to generate figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Methylation, Activation Assay

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 6. Low IGFBP3 expression is associated with a high expression of angiogenic proteins related to HIF-2α under hypoxia. (A) Dot data of angiogenesis antibody array in P0, P4, P4-V, and P4-I lysates under normoxia and hypoxia. IL-8 showed a similar expression pattern as VEGF. Both IL-8 and VEGF were more expressed under hypoxia than in normoxia. (B) Expression signals were presented in categories by importance related to hypoxia and functions related to vasculogenesis. (IL-8 was negatively correlated with HIF-1α and positively correlated with HIF-2α. The proteins listed: IFNα to IL-4 are proteins with anti-angiogenic abilities; angiogenin to VEGF-R3 are known as angiogenic proteins.) The dot signals were analyzed with Image Studio Lite version 5.2, and the relative expression levels were compared between the groups. Red represents a higher signal performance, while green represents a lower signal performance than the comparison cell. Excel 2016 was used to generate chart. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Ab Array, Comparison

Journal: The EMBO Journal

Article Title: IGFBP 1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

doi: 10.15252/embj.201592903

Figure Lengend Snippet: A Schema of the analysis of gene expression in islets isolated from control larvae and larvae subjected to β‐cell ablation. β cells were ablated by exposing nitroreductase (NTR)‐expressing transgenic larvae to metronidazole (MTZ) from 3 to 4 dpf. Islets were then isolated, and their RNA extracted and analyzed by microarray. Out of the 470 genes that were upregulated more than 50%, 33 genes encoded proteins that harbored a signal peptide for secretion (according to the algorithm of SignalP). Excluding genes that encode enzymes or proteins with a transmembrane (TM) domain, we selected 11 genes for overexpression studies in zebrafish larvae (C–E). B Microarray heat map showing the upregulation in expression of the 11 candidate genes in β‐cell‐ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β‐cell ablation. C Schema of the construct used for overexpression of the candidate genes (under the control of the beta‐actin promoter), and expression of GFP in the heart (as an internal control for genomic integration). D, E Representative images at 6 dpf of Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) transgenic larvae that had been injected at the 1–2 cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a ( bactin:igfbp1a ), subjected to β‐cell ablation by metronidazole (MTZ) during 3–4 dpf, and subsequently allowed to regenerate for 2 days. The GFP + heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. F Quantification of β‐cell regeneration at 6 dpf in control ( n = 23), bactin:igfbp1a ‐overexpressing ( n = 13), and bactin:igfbp1b ‐overexpressing ( n = 8) Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) larvae; *** P = 0.0002, ns = non‐significant ( P = 0.3106). G Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg ( ins:GFP ) following β‐cell ablation between 3 and 4 dpf. Scale bar: 50 μm. H Both liver‐specific ( lfabp promoter; n = 46) and widespread ( bactin promoter; n = 18) overexpression of igfbp1a increase β‐cell regeneration when compared to control ( n = 77); *** P < 0.001. I–M Quantification of β cells with or without β‐cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin‐expressing β cells; **** P < 0.0001. Scale bars: 15 μm. Data information: Results are presented as mean values ± SEM and analyzed with one‐way ANOVA (F, H) or two‐way ANOVA (I). See also Fig .

Article Snippet: The islets were then treated with PBS (vehicle control) or 1 μg/ml of recombinant human IGFBP1 protein (R&D Systems) for 72 h. Thereafter, the islets were snap‐frozen in Tissue‐Tek TM (Sakura) and sectioned.

Techniques: Gene Expression, Isolation, Control, Expressing, Transgenic Assay, Microarray, Over Expression, Construct, Injection, Immunohistochemistry, Confocal Microscopy

Journal: The EMBO Journal

Article Title: IGFBP 1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

doi: 10.15252/embj.201592903

Figure Lengend Snippet: A–C Igfbp1a promotes β‐cell regeneration, rather than β‐cell survival. To trace β cells, we exposed Tg ( ins:kaede ) larvae to UV light (which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence) just before ablating the β cells by MTZ treatment from 3 to 4 dpf. After regeneration, the newly formed β cells are green, whereas the β cells that survived ablation are yellow (overlap of green and red). Representative confocal images (A, B) at 6 dpf of control and bactin:igfbp1a ‐overexpressing Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) transgenic larvae; arrows indicate surviving (yellow) β cells. Scale bars: 10 μm. (C) Quantification of β‐cell regeneration (green bars) and β‐cell survival (yellow bars) per larva at 6 dpf. *** P < 0.001; n = 20 larvae in the control group, n = 27 larvae in the bactin:igfbp1a ‐overexpressing group. D–F Igfbp1a does not promote δ‐cell regeneration. We treated control and bactin:igfbp1a ‐overexpressing Tg ( sst:flag‐NTR ) ;Tg ( sst:dsRed ) larvae with MTZ from 3 to 4 dpf to ablate δ cells, and then allowed them to regenerate until 6 dpf. Representative confocal images (D, E) at 6 dpf of control and bactin:igfbp1a ‐overexpressing larvae showing comparable number of δ cells after 2 days of regeneration. Scale bars: 15 μm. (F) Quantification of the total number of δ cells per δ‐cell‐ablated larva at 6 dpf compared to the baseline number of δ cells in non‐ablated control larvae. ns, P = 0.2325; n = 13 in the control group, n = 7 in the bactin:igfbp1a group. G Other Igfbps do not promote β‐cell regeneration. We injected Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) transgenics with either transposase mRNA (control; n = 31) or transposase mRNA + one of six different igfbps , igfbp2a ( n = 25), igfbp2b ( n = 25), igfbp3 ( n = 26), igfbp5a ( n = 17), igfbp6b ( n = 34), and igfbp7 ( n = 17), treated them with MTZ from 3 to 4 dpf to ablate their β cells, and then quantified their β cells after 2 days of regeneration, at 6 dpf; ns=non‐significant (igfbp2a P = 0.986, igfbp3 P = 0.979, igfbp5a P = 0.999, igfbp6b P = 0.997, and igfbp7 P = 0.999); igfbp2b * P = 0.037. H Tg ( pcsk1:GFP ) is expressed in regenerating β cells within the islet (arrowheads), but not outside the islet (arrow), at 6 dpf in larvae overexpressing igfbp1a . Scale bar: 15 μm. I, J Free‐glucose levels during β‐cell regeneration in control ( bactin:mCherry ) and bactin : igfbp1a ‐overexpressing Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) larvae (I), as well as in Tg ( ins:kaede ) ;Tg ( ins:CFP‐NTR ) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3 to 4 dpf to ablate their β cells and monitored their free‐glucose levels at 3–7 dpf. Free‐glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1‐protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β‐cell ablation. n = 24 larvae (four pools of six larvae) per data point; *** P < 0.001, * P < 0.05. Data information: Results are presented as mean values ± SEM and analyzed with t ‐test (C), Mann‐Whitney test (F), one‐way ANOVA (G), or two‐way ANOVA (I, J).

Article Snippet: The islets were then treated with PBS (vehicle control) or 1 μg/ml of recombinant human IGFBP1 protein (R&D Systems) for 72 h. Thereafter, the islets were snap‐frozen in Tissue‐Tek TM (Sakura) and sectioned.

Techniques: Fluorescence, Control, Transgenic Assay, Injection, Recombinant, Over Expression, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: IGFBP 1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

doi: 10.15252/embj.201592903

Figure Lengend Snippet: Isolated islets from mice and from human donors were treated for 3 days with recombinant mouse or human IGFBP 1 protein, respectively. A, B Representative confocal images of control and Igfbp1‐treated mouse islets. gcg + ins + cells are indicated by arrows. Scale bars: 25 μm. C Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in mouse islets after treatment with increasing concentrations of Igfbp1. P = 0.7304 (Igfbp1 0.3 μg/ml), ** P = 0.0023 (1 μg/ml), ** P = 0.0013 (10 μg/ml), * P = 0.0410 (25 μg/ml), respectively. Multiple optical sections from 10–50 islets per group, from 4–8 different mice, were used for the quantifications. D Quantification of β‐cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared. Multiple optical sections from 14–22 islets, from 3 different mice, were used for the quantifications. E, F Representative confocal images of control or IGFBP1‐treated human islets. A gcg + ins + cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. G Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in human islets from five donors. ** P = 0.0036. Co‐expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa. ** P < 0.0028; n = 115 sections in the control group, n = 147 sections in the IGFBP1 group. H Our proposed mechanism by which the extracellular signals insulin, IGFs, and IGFBP1 can regulate α‐ to β‐cell transdifferentiation. Data information: Results are presented as mean values ± SEM and analyzed with Kruskal‐Wallis (C) or Mann‐Whitney tests (D, G). Source data are available online for this figure.

Article Snippet: The islets were then treated with PBS (vehicle control) or 1 μg/ml of recombinant human IGFBP1 protein (R&D Systems) for 72 h. Thereafter, the islets were snap‐frozen in Tissue‐Tek TM (Sakura) and sectioned.

Techniques: Isolation, Recombinant, Control, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: IGFBP 1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

doi: 10.15252/embj.201592903

Figure Lengend Snippet: A–D Correlations between baseline levels of fasting IGFBP1 levels and BMI (A, C) or fasting insulin levels (B, D) in men (A, B) and women (C, D). The correlations between these baseline values are significant both in subjects who developed T2D (red squares) and in controls with a normal glucose tolerance (NGT) (blue circles) at follow‐up after 8–10 years. Results are presented as individual values with linear regression. Related to Table .

Article Snippet: The islets were then treated with PBS (vehicle control) or 1 μg/ml of recombinant human IGFBP1 protein (R&D Systems) for 72 h. Thereafter, the islets were snap‐frozen in Tissue‐Tek TM (Sakura) and sectioned.

Techniques:

Journal: The EMBO Journal

Article Title: IGFBP 1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

doi: 10.15252/embj.201592903

Figure Lengend Snippet: High levels of IGFBP1 are associated with a lower risk of developing type‐2 diabetes

Article Snippet: The islets were then treated with PBS (vehicle control) or 1 μg/ml of recombinant human IGFBP1 protein (R&D Systems) for 72 h. Thereafter, the islets were snap‐frozen in Tissue‐Tek TM (Sakura) and sectioned.

Techniques:

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 2 BMP2 increases IGFBP3 production in HTR8/SVneo and primary human EVT cells. A and B, HTR8/SVneo cells (left panel, N = 3) and primary EVT cells (right panel, N = 5) were treated with or without 25 ng/mL BMP2 for different lengths of time (3, 6, 12, or 24 hours). A, IGFBP3 mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. B, IGFBP3 protein levels were examined by western blot and normalized to α-tubulin. C, HTR8/SVneo cells (left panel) or primary EVT cells (right panel) were treated with or without 25 ng/mL BMP2 for 12 or 24 hours. IGFBP3 accumulation in conditioned medium was measured using an enzyme immunoassay. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < .05)

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 3 ID1 mediates BMP2-induced IGFBP3 upregulation in human trophoblast cells. A and B, HTR8/SVneo cells were transfected for 48 hours with 25 nM nontargeting control siRNA (siCtrl) or 25 nM siRNA targeting ID1 (siID1) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2. A, RT-qPCR was used to measure IGFBP3 and ID1 mRNA levels 12 hours after treatment with BMP2 (N = 3). B, Western blot was used to measure IGFBP3 and ID1 protein levels 24 hours after BMP2 treatment (N = 4). C, Primary EVT cells were transfected for 48 hours with 25 nM siCtrl or siID1 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for another 24 hours. Western blot was used to measure IGFBP3 and ID1 protein levels (N = 5). Summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < .05)

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 4 Knockdown of ID1 or IGFBP3 abolishes BMP2-induced trophoblast cell invasion in HTR8/SVneo cells (N = 3). A-C, HTR8/ SVneo cells were transfected for 48 hours with 25 nM siCtrl, siID1, or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 36 hours. Knockdown efficiency of ID1 and IGFBP3 were evaluated by western blot. D and E, HTR8/SVneo cell invasiveness was examined using a Matrigel-coated transwell invasion assay and combined quantitative results are shown below representative images of the invasion assays. Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < .05)

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques: Knockdown, Transfection, Western Blot, Transwell Invasion Assay

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 5 Knockdown of ID1 or IGFBP3 abolishes the BMP2-induced trophoblast cell invasion in primary human EVTs (N = 5). A-C, Primary human EVTs were transfected for 48 hours with 25 nM siCtrl, siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 36 hours. Knockdown efficiency of ID1 and IGFBP3 were evaluated by western blot. D and E, Primary human EVT cell invasiveness was examined by Matrigel-coated transwell invasion assay and summarized quantitative results are shown in E. Results are presented as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < .05)

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques: Knockdown, Transfection, Western Blot, Transwell Invasion Assay

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 6 ID1 and IGFBP3 are involved in BMP2-induced endothelial-like tube formation and PlGF secretion in HTR8/SVneo cells (N = 3). A, HTR8/SVneo cells were transfected for 48 hours with 25 nM siCtrl, siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 24 hours. Left panels show representative images from the endothelial-like tube formation assays. Right panel shows combined quantitative results from image analysis of total tube length. B, HTR8/SVneo cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, or 12 hours). PlGF mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. C, HTR8/SVneo cells were transfected for 48 hours with 25 nM siCtrl, siID1 or siIGFBP3 prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 24 hours. PLGF accumulation in conditioned medium was measured using an enzyme immunoassay. Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < .05)

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques: Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial‐like tube formation through ID1‐mediated upregulation of IGF binding protein‐3

doi: 10.1096/fj.201902168rr

Figure Lengend Snippet: FIGURE 7 Proposed model for the involvement of ID1 and IGFBP3 in BMP2-induced human trophoblast invasion and endothelial-like tube formation. BMP2 binds to type I and II receptors leading to ID1-mediated IGFBP3 upregulation. Increased ID1 and IGFBP3 contribute to BMP2-induced human trophoblast invasion, possibly via.ID1- and IGFBP3-mediated induction of the EMT- associated transcription factor SLUG. Increased ID1 and IGFBP3 also contribute to BMP2-induced human trophoblast endothelial-like tube formation and PlGF secretion, and the latter may be essential for the pro-angiogenic effects of BMP2

Article Snippet: Mouse monoclonal anti-IGFBP3 antibody (catalog no. MAB305) was purchased from R&D Systems.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: IGFBP7 is expressed at low levels in CRC tumor vascular endothelial cells and correlates with poor prognosis. A Schematic diagram of the experimental design for scRNA-seq and NuGEN RNA sequencing. Created with BioRender.com. B UMAP plots showing cell resources from two different tissue types. C UMAP plots showing cell annotations from two tissues. D Marker genes for each cell type. E Expression levels of IGFBP7 in each cell type. F Expression levels of endothelial IGFBP7 in adenoma and adenocarcinoma tissues. G NuGEN RNA sequencing results showing the expression levels of endothelial IGFBP7 in CD31+ cells. H Representative RNAscope image showing IGFBP7 expression in endothelial cells of three paired CRC tumors and para-tumor tissues (scale bar, 50 μm). I IGFBP7 expression levels in endothelial cells from paired CRC tumors and para-tumor tissues. J Kaplan-Meier analysis of the association between IGFBP7 expression levels in endothelial cells and overall survival in CRC patients. K Kaplan-Meier analysis of the association between IGFBP7 expression levels in endothelial cells and disease-free survival in CRC patients. L Cox analysis of the association between IGFBP7 expression levels in endothelial cells and disease-free survival in CRC patients. M Proportion of IGFBP7 in endothelial cells from paired CRC tumors and para-tumor tissues and representative flow cytometry plots. N IGFBP7 expression levels in the serum of CRC patients and healthy controls

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: RNA Sequencing, Marker, Expressing, RNAscope, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: Endothelial IGFBP7 expression is downregulated in a stage-specific manner during inflammation-driven carcinogenesis. A Schematic diagram showing the timeline for mouse euthanasia at different time points in the AOM/DSS-induced colitis-associated CRC model. Created with BioRender.com. B PCA analysis of transcriptome sequencing in mouse colon tissue. C Representative immunofluorescence images of IGFBP7 expression in endothelial cells of mice at different time points (scale bar, 50 μm). D , E Fluorescence intensity statistics of IGFBP7 expression in endothelial cells of mice at different time points. F , G Flow cytometry analysis statistics of IGFBP7 expression in endothelial cells of mice at different time points. H Pseudo-time analysis of AOM/DSS model mouse colon tissue from T1 to T4 stages based on Mfuzz. I Enrichment analysis of genes in Cluster 7. J Schematic representation of the AOM/DSS-induced colitis-associated colorectal cancer model in Igfbp7fl/fl; Cdh5-Cre+/− mice. Created with BioRender.com. K Colon length of Igfbp7fl/fl; Cdh5-Cre+/− mice following AOM/DSS treatment. L Tumor number in the colon of Igfbp7fl/fl; Cdh5-Cre+/− mice following AOM/DSS treatment

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: Expressing, Sequencing, Immunofluorescence, Fluorescence, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: IGFBP7 secreted by endothelial cells is internalized by tumor cells. A IGFBP7 mRNA expression levels in endothelial cell lines, CRC cell lines and FHC. B Secretion levels of IGFBP7 in the culture media supernatant from endothelial cell lines, CRC cell lines and FHC. C IGFBP7 protein expression levels in endothelial cell lines, CRC cell lines and FHC. D Changes in IGFBP7 secretion after treating HMEC-1 cells with Golgi Stop (1 µl/ml) or GW4869 (10 µM) for 6 h. E Schematic diagram of co-culture between endothelial cells and CRC cells for 48 h. Created with BioRender.com. F Changes in IGFBP7 protein levels after co-culturing CRC and HMEC-1 for 48 h. G Changes in IGFBP7 mRNA levels after co-culturing CRC cells and HMEC-1 for 48 h. H , I Changes in IGFBP7 secretion levels in the culture media supernatant after co-culturing CRC cells and HMEC-1 for 48 h. J Detection of IGFBP7 protein levels after 48 h co-culture of CRC cells and HMEC-1, with MG132 (20 µM) or DMSO added during the final 5 h. K , L Detection of IGFBP7 mRNA expression levels in CRC tumor cells after treatment with recombinant IGFBP7 protein (100, 300, or 500 ng/ml) for 48 h. M Detection of IGFBP7 and 6x His-tag protein expression levels in CRC tumor cells after treatment with recombinant IGFBP7 protein (100, 300, or 500 ng/ml) for 48 h

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: Expressing, Co-Culture Assay, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: Endothelial-derived IGFBP7 suppresses colorectal cancer cell migration and proliferation via TGF-β1 signaling. A Venn diagram showing the intersection of enriched GSEA pathways in transcriptome sequencing results of SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h. B GSEA enrichment plot of the “GO_BP_CELL_MIGRATION” pathway in tumor cells after co-culture with recombinant IGFBP7 protein (100 ng/ml) for 48 h. C GSEA enrichment plot of the “GO_BP_BLOOD_VESSEL_MORPHOGENESIS” pathway in tumor cells after co-culture with recombinant IGFBP7 protein (100 ng/ml) for 48 h. D Venn diagram showing the intersection of downregulated genes enriched in the “GO_BP_CELL_MIGRATION” pathway (fc < 0, p < 0.05) identified from transcriptome sequencing of SW480, HCT116, and CACO2 tumor cell lines co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h. E Heatmap showing the common downregulated genes in tumor cell lines, with data sourced from SW480. F , G The mRNA expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA in tumor cell lines after co-culture with endothelial cells for 48 h. H The protein expression levels of TGF-β1, E-cadherin, Vimentin, and PCNA in tumor cell lines after co-culturing with endothelial cells for 48 h. I SW480 cells were transfected with plasmids and co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 24 h, followed by measurement of luciferase activity. Data were normalized to Renilla luciferase activity (firefly/Renilla) and are presented as fold change relative to the control group (control = 1). J SW480 cells were transfected with an EGR1 overexpression plasmid and co-cultured with recombinant IGFBP7 protein (100 ng/ml) for 48 h, followed by analysis of EGR1, TGF-β1, and IGFBP7 expression levels. K , L , M Cell migration assays were performed to assess the migration of SW480 and HCT116 tumor cells treated for 48 h with the culture medium supernatant from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA (scale bar, 200 μm). N , O , P Scratch wound healing assays were performed to evaluate the healing of SW480 and HCT116 tumor cells treated for 24 h with the culture medium supernatant from HMEC-1 or HUVEC endothelial cells transiently transfected with IGFBP7 siRNA (scale bar, 100 μm). Q , R CCK-8 cell proliferation assays were performed to assess the proliferation of SW480 and HCT116 tumor cells treated for 24, 48, 72, and 96 h with the culture medium supernatant from HMEC-1 cells transiently transfected with IGFBP7 siRNA. S , T CCK-8 cell proliferation assays were performed to assess the proliferation of SW480 and HCT116 tumor cells treated for 24, 48, 72, and 96 h with the culture medium supernatant from HUVEC cells transiently transfected with IGFBP7 siRNA

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: Derivative Assay, Migration, Sequencing, Cell Culture, Recombinant, Co-Culture Assay, Expressing, Transfection, Luciferase, Activity Assay, Control, Over Expression, Plasmid Preparation, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: Loss of IGFBP7 enhances angiogenic potential of endothelial cells. A Hierarchical clustering dendrogram of coexpression modules identified using WGCNA following NuGEN RNA sequencing of endothelial cell sorting. B Dot plot illustrating the enriched pathway of IGFBP7 containing network. C GSEA enrichment plot of the “GOBP_REGULATION_OF_VASCULATURE_DEVELOPMENT” pathway in endothelial cells following transient transfection with IGFBP7 siRNA. D Heatmap of transcriptome data following transient transfection of IGFBP7 siRNA in endothelial cells. Red indicates upregulated genes, blue indicates downregulated genes, and the intensity of the color reflects the degree of expression change. E , F mRNA level validation of angiogenesis-related genes following transient transfection of IGFBP7 siRNA in endothelial cells. G , H , I Angiogenesis assays were performed to assess the angiogenic ability of endothelial cells following transient transfection with IGFBP7 siRNA. HMEC-1 cells (2 × 10⁴ cells/well) or HUVECs (5 × 10³ cells/well) transfected with siRNA were seeded onto Matrigel and incubated at 37 °C for 6 h (scale bar, 100 μm)

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: RNA Sequencing, FACS, Transfection, Expressing, Biomarker Discovery, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: IGFBP7 inhibits tumor growth and angiogenesis in mouse models. A Schematic diagram illustrating the experimental design of intraperitoneal injection of saline (100 µl/mouse), recombinant IGFBP7 protein (10 µg/mouse), IgG (100 µg/mouse), or anti-mouse IGFBP7 antibody (100 µg/mouse) in the subcutaneous tumor mouse model. Created with BioRender.com. B , C Representative tumor images of subcutaneous tumors from mice in different treatment groups. D Quantification of tumor weight in different treatment groups. E , F Tumor growth curves showing tumor volume in mice from different treatment groups. G Representative immunohistochemistry (IHC) staining images showing the expression of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) in mouse tumor tissues. H Bar graph displaying the optical density values of CD31-positive areas in tumor tissues from different treatment groups, reflecting changes in angiogenesis. I Bar graph displaying the optical density values of TGF-β1-positive areas in tumor tissues from different treatment groups, reflecting TGF-β1 expression levels. J Schematic diagram illustrating the experimental design of intraperitoneal injection of saline (100 µl/mouse), recombinant IGFBP7 protein (10 µg/mouse), IgG (100 µg/mouse), or anti-mouse IGFBP7 antibody (100 µg/mouse) in the AOM/DSS-induced colitis-associated colorectal cancer model. Created with BioRender.com. K , L Representative images of colorectal tumors in AOM/DSS-induced colitis-associated CRC model from different treatment groups. M Quantification of tumor number in AOM/DSS-induced colitis-associated CRC model from different treatment groups. N Representative immunohistochemistry (IHC) staining images showing the expression of CD31 (scale bar, 100 μm) and TGF-β1 (scale bar, 75 μm) in colorectal tumor tissues from the AOM/DSS-induced colitis-associated CRC model. O Bar graph displaying the vascular density of CD31-positive areas in tumor tissues from different groups. P Bar graph displaying the optical density values of TGF-β1-positive areas in tumor tissues from different groups. Q Schematic diagram illustrating the experimental design in the AOM/DSS-induced colitis-associated colorectal cancer model. Mice were administered intraperitoneal injections of saline (100 µl/mouse) or recombinant IGFBP7 protein (10 µg/mouse) once per week, as well as mouse IgG2a Kappa antibody (5 mg/kg) or the anti-VEGF monoclonal antibody B20-4.1.1.1 (5 mg/kg) twice per week. Created with BioRender.com. R , S Representative images and quantitative analysis of colon length in mice. T , U Representative images and quantitative analysis of the number of colonic tumors in mice

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: Injection, Saline, Recombinant, Immunohistochemistry, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Endothelial cell-derived IGFBP7 suppresses angiogenesis and tumor progression in colorectal cancer via the VAPA-TGF-β1 pathway

doi: 10.1186/s13046-026-03664-z

Figure Lengend Snippet: VAPA facilitates lysosomal degradation of IGFBP7 and modulates TGF-β1 signaling. A Immunoprecipitation-mass spectrometry (IP-MS) results showing the proteins that interact with IGFBP7. B Immunoprecipitation analysis demonstrating the interaction between endogenous IGFBP7 and VAPA in HCT116cells. C Immunoprecipitation analysis demonstrating the interaction between endogenous VAPA and IGFBP7 in CaCO2cells. D , E Validation of protein levels after VAPA knockdown in tumor cells. F Protein expression levels of IGFBP7 after co-culturing VAPA knockdown tumor cells with endothelial cells for 48 h. G Changes in the concentration of IGFBP7 in the culture medium after co-culturing VAPA knockdown tumor cells with recombinant IGFBP7 protein (100 ng/ml) for 48 h. H Measurement of IGFBP7 protein expression levels in VAPA -knockdown SW480 cells with or without co-culture with recombinant IGFBP7 (100 ng/ml) for 48 h, followed by treatment with Bafilomycin A1 (20 µM) during the final 6 h. I Measurement of lysosome counts in VAPA knockdown SW480 cells after treatment with recombinant IGFBP7 protein (100 ng/ml) for 48 h. J Representative immunofluorescence staining of IGFBP7 and LAMP1 in HCT116 after VAPA knockdown or co-culturing with recombinant IGFBP7 protein (100 ng/ml) for 48 h. Green, IGFBP7; Red, LAMP1; Blue, nucleus (scale bars, 50 μm). K Relative fluorescence intensities of IGFBP7 from the indicated groups were quantified. L Relative fluorescence intensities of LAMP1 from the indicated groups were quantified. M Protein expression levels of IGFBP7, TGF-β1 signaling pathway, and EMT pathway markers after co-culturing VAPA knockdown SW480 tumor cells with HMEC-1 endothelial cells for 48 h

Article Snippet: Tumor cells were treated with recombinant IGFBP7 protein (R&D Systems, Cat# 1334-B7) for 48 h. After treatment, the culture supernatants and cells were collected for subsequent experiments.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Biomarker Discovery, Knockdown, Expressing, Concentration Assay, Recombinant, Co-Culture Assay, Immunofluorescence, Staining, Fluorescence

Journal: Haematologica

Article Title: RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia

doi: 10.3324/haematol.2022.280757

Figure Lengend Snippet: Detection of the expression levels of potential RHOA downstream targets in chronic myeloid leukemia cells and primary patients’ samples. (A) Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis was used to verify differential expression of several of the most highly downregulated and upregulated genes in K562 RHOA knockout (KO) cells compared with mock control (MC) cells (N=3). Analysis of the same genes in the KU812 RHOA KO cells using RT-qPCR showed the same differential expression patterns. (B) The reduced expression levels for IGFBP2, IL20RB and CD24 were further confirmed using western blotting. (C) Analysis of the GSE47927 chronic myeloid leukemia (CML) dataset revealed a highly significant difference in IGFBP2 expression in CML samples (n=52) compared with normal controls (n=15). When cells from these CML cases were sub-fractionated into different stem/progenitor subpopulations, there was a highly significant increase in IGFBP2 expression in CML hematopoietic stem cells compared with normal counterparts. In similar comparisons, a significant increase in IGFBP2 expression was seen in common myeloid progenitors and megakaryocyte-erythroid progenitors but no difference was seen in granulocyte-monocytic progenitors. Statistical significance was established using the Student t test. ** P ≤0.001, *** P ≤0.0001, **** P ≤0.00001. HSC: hematopoietic stem cells; CMP: common myeloid progenitors; GMP: granulocyte-monocytic progenitors; MEP: megakaryocyte-erythroid progenitors.

Article Snippet: For the IGFBP2 rescue experiment, 100 ng/mL recombinant human IGFBP2 protein (R&D Systems, #674-B2-025) were added to the growth medium prior to the cell adhesion and migration assays.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Quantitative Proteomics, Knock-Out, Control, Western Blot

Journal: Haematologica

Article Title: RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia

doi: 10.3324/haematol.2022.280757

Figure Lengend Snippet: RHOA activates IFGBP2 expression through serum response factor. (A) Luciferase expression analysis shows that in the presence of the IGFBP2 promoter there was an increase in activity that was proportional to the levels of serum response factor (SRF) transfected in the same cells. (B) When SRF was overexpressed in either NIH3T3 or BaF3 cells, there was a proportional increase in IGFBP2 expression. (C) When K562 cells were treated with RHOA activator I, levels of SRF declined in the cytoplasm but increased in the nucleus. The relative purity of the cytoplasmic and nuclear protein enrichment was demonstrated by the almost exclusive presence of proteins GAPDH (cytoplasm) and Lamin A (nucleus). (D) The same increased levels of SRF in the nucleus of adherent 3T3 cells following RHOA activation was seen using confocal microscopy. (E) Chromatin immunoprecipitation quantitative polymerase chain reaction analysis from K562 cells using primers P1, which target the IGFBP2 promoter region, showed occupancy of SRF on the IGFBP2 promoter (SRF-), which was increased when the cells were treated with the RHOA activator (SRF+). In the same experimental model, no significant changes were seen in the downstream intron region defined by the P2 primers. The scale bar in (D) represents 50 µm. Differences between the knockout and matched control cells were evaluated using the Student t test. Cells transduced with empty vector were used as the control for comparison in (B). * P <0.01, ** P ≤0.001, *** P ≤0.0001, **** P ≤0.00001, ns: not significant. MSCV: murine stem cell virus; EV: empty vector.

Article Snippet: For the IGFBP2 rescue experiment, 100 ng/mL recombinant human IGFBP2 protein (R&D Systems, #674-B2-025) were added to the growth medium prior to the cell adhesion and migration assays.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Protein Enrichment, Activation Assay, Confocal Microscopy, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Knock-Out, Control, Transduction, Plasmid Preparation, Comparison, Virus

Journal: Haematologica

Article Title: RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia

doi: 10.3324/haematol.2022.280757

Figure Lengend Snippet: Knockout of IFGBP2 partially recapitulates the phenotype of RHOA loss. (A) Analysis of the supernatant from K562 C8 and KU812 C7 knockout (KO) cells when compared with the respective mock control (MC) cells, showed a dramatic reduction in IGFBP2 protein levels in the KO cell culture medium. (B) In migration and adhesion analysis, K562 KO C8 cells showed a reduction in both phenotypes compared with MC cells. When exogenous, recombinant IGFBP2 (rIGFBP2) was added to the culture medium, there was a significant increase in both phenotypes in the KO C8 cells. (C, D) CRISPR knockout of IGFBP2 in K562 cells generated two clones, C5 and C11 (C), which when subjected to migration and adhesion assays (D) showed reduced levels compared with MC cells. (E) When these cells were xenografted into NSG hosts, mice receiving KO clones C5 and C11 showed an increased survival compared with MC-engrafted mice. (F) This result was consistent with luminescence intensity in the mice after 28 days, which showed that the tumor burden was significantly reduced in the mice grafted with the KO C5 cells compared to that of mice engrafted with MC cells. White blood cell count at the time of sacrifice was also reduced in the mice grafted with KO C5 cells. The scale bars in (B) and (D) represent 400 µm, with the magnification for the images in (B) and (D) being the same. Differences between the KO and MC cells were evaluated using the Student t test. Pairwise comparisons are indicated by the horizontal lines. ** P ≤0.001, *** P ≤0.0001, **** P ≤0.00001. CM: culture medium; WBC: white blood cells.

Article Snippet: For the IGFBP2 rescue experiment, 100 ng/mL recombinant human IGFBP2 protein (R&D Systems, #674-B2-025) were added to the growth medium prior to the cell adhesion and migration assays.

Techniques: Knock-Out, Control, Cell Culture, Migration, Recombinant, CRISPR, Generated, Clone Assay, Cell Counting

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: IGFBP6 was detected by immunostaining in primary NPC tissues (magnification ×200). Left:, IGFBP6 positive staining; Middle: IGFBP6 negative staining; Right: isotype control staining ( A ) IGFBP6 mRNA was measured in five NPC cell lines (CNE2, CNE1, SUNE1, HK1 and HONE1) via RT-PCR, with GAPDH as an internal control ( B ) Data are representative of three separate experiments. Western blotting of whole-cell lysates to detect IGFBP6 ( C ) IGFBP6 levels in CM from NPC cells as measured by ELISA ( D ) Data are representative of two separate experiments. All data represent means ± SD from triplicates.

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Immunostaining, Staining, Negative Staining, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: Correlation of IGFBP6 expression with clinical characteristics in patients with NPC

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: Multivariate Cox regression analysis for survival prognostic factors in advanced nasopharyngeal carcinoma

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: CNE2 (upper panel) and HK1 (lower panel) cell proliferation as measured by MTS assay ( A ) Data represent means ± SD from six wells. * P < 0.05 compared to controls (IGFBP6 0 ng/ml). In transwell assays (upper panel), exogenous IGFBP6 inhibited CNE2 and HK1 cell invasion compared to controls ( B ) Invasive Index (%) was calculated (lower panel) according to the manufacturer's instructions. Columns, means of triplicate assays; bars, SE. ** P < 0.01 compared to controls.

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: MTS Assay

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: CNE2 cells were stably transfected with IGFBP6-shRNA. Real-time RT-PCR confirmed knockdown efficiency ( A ) Bars, ± SE. Data are representative of three separate experiments. Western blotting confirmed IGFBP6 knockdown ( B ) IGFBP6 knockdown induced tumor cell proliferation compared to controls ( C ) Representative wound-healing assay images ( D ) IGFBP6 knockdown increased tumor cell migration ( E ) Data represent means ± SD. * P < 0.05 compared to controls. Western blotting revealed GSK3β/β-catenin/cylin D1 pathway activation as a result of IGFBP6 knockdown ( F ).

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Stable Transfection, Transfection, shRNA, Quantitative RT-PCR, Knockdown, Western Blot, Wound Healing Assay, Migration, Activation Assay

Journal: Oncotarget

Article Title: IGFBP6 is a novel nasopharyngeal carcinoma prognostic biomarker

doi: 10.18632/oncotarget.11886

Figure Lengend Snippet: Silencing IGFBP6 expression in CNE2 cells promotes tumor metastasis in a mouse model

Article Snippet: Recombinant human IGFBP6 (rhIGFBP6) and the anti-human IGFBP6 monoclonal antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing