recombinant human igfbp3  (R&D Systems)

Journal: Journal of Proteome Research

Article Title: Comparative Tear Fluid Proteomics to Explore Treatment Responsiveness in Diabetic Macular Edema: A Pilot Study

doi: 10.1021/acs.jproteome.5c00181

Figure Lengend Snippet: Enzyme-linked immunosorbent assay (ELISA) data of the targeted tear fluid protein samples. A. TIMP-1, MMP-9, and IGFBP3 levels (ng/mL) in different groups. B. The principal component analysis (PCA) plot using bar plot data. The arrow indicates the change of IGFBP3 to MMP-9 ratio was most associated with group difference (treatment responsiveness in this study). C. The ratios of TIMP-1/MMP-9 and IGFBP3/MMP-9 following treatment across various groups. D. Changes in the above ratios, calculated as the difference between post-treatment and pretreatment values, across the same groups. Asterisk (*) denotes statistical significant difference between groups ( p < 0.05).

Article Snippet: Metallopeptidase inhibitor 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and insulin-like growth factor-binding protein 3 (IGFBP3) were tested according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: TMEM219 signaling promotes intestinal stem cell death and exacerbates colitis

doi: 10.1172/jci185783

Figure Lengend Snippet: Figure 3. Mechanistic studies delineate a TMEM219-related proapoptotic downstream signaling. (A and B). Confocal microscopy analysis (Scale bar: 10 μm; original magnification ×63) depicting colocalization and binding of TMEM219 (green) and IGFBP3 (red) in intestinal cells dissociated from a control sample and incubated with recombinant IGFBP3 overnight and in CaCo2 cells. Cells were stained with DAPI for nuclei (blue) and immunolabeled with anti-TMEM219 (green) and anti-IGFP3 Abs (red). (C). Cleaved/activated Caspases 1, 2, 3, 7, 8, and 9 were quantified in CaCo2 cells cultured with/without IGFBP3 (50 ng/mL) and with/without the TMEM219 inhibitor ecto-TMEM219 (newly generated recombinant protein based on the TMEM219 extracellular portion). (D). Cell death quantified in CaCo2 cells cultured with/without IGFBP3 with ecto-TMEM219, Pan-caspase inhibitor, and selective inhibitors for Caspases 1, 3, 7, 8, and 9. (E and F). Phosphoproteomic profile identified in CaCo2 cells cultured with/without IGFBP3 (50 ng/mL) and with/without ecto-TMEM219 (130 ng/mL). Differ- entially expressed phosphorylated proteins (normalized to control) are presented in the heatmap as a ratio between nonphosphorylated and phosphorylated protein (mean value). In F, up/downregulated phosphorylated proteins with IGFBP3 and with Ecto-TMEM219 are reported. (G and H). Development of miniguts obtained from crypts of patients with active Crohn’s disase (CD) and cultured with/without IGFBP3 and ecto-TMEM219 (n = 7). Original magnification ×20; scale bar: 100 μm. (I and J). Normalized mRNA expression of EPHB2 (I) and LGR5 (J) in miniguts as described in G (n = 6). (K and L). Development of miniguts obtained from crypts of controls (Ctrl) and cultured in the presence of pooled sera of patients with active CD in place of 10% FBS and with/without ecto- TMEM219 (n = 7). Original magnification ×20; scale bar: 100 μm. (M and N). Normalized mRNA expression of EPHB2 (M) and LGR5 (N) in miniguts obtained as reported in K (n = 5). Mean ± SEM. At least 3 independent experiments run in duplicate. 1-way ANOVA followed by Šidák’s post hoc test and 2-sided t test.

Article Snippet: Cleaved Caspase 8 and phosphorylated-AKT were assessed using ELISA (MBS766157, MyBiosource and KHO0111, Invitrogen) in human crypts, patient-derived organoids, and CaCo2 cells cultured with/without IGFBP3 (50 ng/mL, 8874-B3, R&D Systems), with or without ecto-TMEM219 (130 ng/mL, Genscript) (22).

Techniques: Confocal Microscopy, Binding Assay, Control, Incubation, Recombinant, Staining, Immunolabeling, Cell Culture, Generated, Expressing

Journal: Journal of Clinical Investigation

Article Title: TMEM219 signaling promotes intestinal stem cell death and exacerbates colitis

doi: 10.1172/jci185783

Figure Lengend Snippet: Figure 4. Pharmacological blockade of IGFBP3/TMEM219 signal ameliorates DSS-mediated acute and chronic colitis in vivo. (A). Experimental design of the DSS acute prevention model. (B–D). Disease activity index (DAI), histological score, and colon length measured at day +12 in control (n = 5), DSS+PBS and DSS+ecto-TMEM219–treated mice (n = 7–10). (E). Representative pictures of H&E staining, crypt proliferation (MKI67), and ALDH immunostaining in colons of controls, DSS+PBS, and DSS+ecto-TMEM219–treated mice. Original magnification ×20 (upper and middle panels), ×40 (lower panels); scale bars: 100 μm. Arrows indicate ALDH+ cells (lower panels). (F and G). Development of 8-day miniguts obtained from colons of controls, DSS+PBS, and DSS+ecto- TMEM219–treated mice (n = 5/group). Original magnification ×20; scale bar: 100 μm. (H). Experimental design of the DSS chronic treatment model. (I–K). DAI (n = 10), histological score (n = 5), and colon length (n = 10) measured at day 42 in controls, DSS+PBS, and DSS+ecto-TMEM219–treated mice. (L). Representative pictures of H&E staining, crypt proliferation (MKI67), and ALDH immunostaining in colons of controls, DSS+PBS, and DSS+ecto-TMEM219– treated mice. Original magnification ×20 (upper and middle panels) and ×40 (lower panels); scale bar: 100 μm. Arrows indicate MKI67+ cells (middle panel) and ALDH+ cells (lower panels). (M and N). Colon endoscopic analysis in controls, in mice receiving DSS+PBS (n = 9),or DSS+ecto-TMEM219 (n = 6), (Day 42). Mean ± SEM. 1-way ANOVA followed by Šidák’s post hoc test and 2-sided t test.

Article Snippet: Cleaved Caspase 8 and phosphorylated-AKT were assessed using ELISA (MBS766157, MyBiosource and KHO0111, Invitrogen) in human crypts, patient-derived organoids, and CaCo2 cells cultured with/without IGFBP3 (50 ng/mL, 8874-B3, R&D Systems), with or without ecto-TMEM219 (130 ng/mL, Genscript) (22).

Techniques: In Vivo, Activity Assay, Control, Staining, Immunostaining

Journal: Journal of Clinical Investigation

Article Title: TMEM219 signaling promotes intestinal stem cell death and exacerbates colitis

doi: 10.1172/jci185783

Figure Lengend Snippet: Figure 5. Pharmacological blockade of the IGFBP3/TMEM219 signaling improves colitis in vivo in the T cell transfer model. (A). Experimental design of the T cell–mediated acute colitis prevention model, in which mice received T cells at day 0 and developed colitis between days 21 and 35 and were administered treatment with ecto-TMEM219 or with the positive control anti-p40 compound, from day 14 to day 42. (B). Percentage weight change in mice subjected to T cell–induced colitis and treated with ecto-TMEM219, anti-p40, or PBS (n = 8). (C and D). Colitis score and representative endoscopic pictures obtained at day 42 in naive mice (RAG–/–), in RAG–/– mice receiving T cells plus PBS, T cells plus anti-p40, or ecto-TMEM219 in the T cell transfer model (n = 6–10). (E). Rep- resentative pictures of histological analysis in colon samples of naive mice, mice receiving T cells plus PBS, T cells plus anti-p40, or ecto-TMEM219. Original magnification ×20; scale bar: 100 μm. (F and G). Histological score and colon length measured at day 42 in naive mice (n = 5) and in mice receiving T cells + PBS or T cells + anti-p40 or ecto-TMEM219 in the T cell transfer model (n = 8–12). Mean ± SEM. 2-way or 1-way ANOVA followed by Šidák’s post hoc test and 2-sided t test, Mann-Whitney t test.

Article Snippet: Cleaved Caspase 8 and phosphorylated-AKT were assessed using ELISA (MBS766157, MyBiosource and KHO0111, Invitrogen) in human crypts, patient-derived organoids, and CaCo2 cells cultured with/without IGFBP3 (50 ng/mL, 8874-B3, R&D Systems), with or without ecto-TMEM219 (130 ng/mL, Genscript) (22).

Techniques: In Vivo, Positive Control, MANN-WHITNEY

Journal: Journal of Clinical Investigation

Article Title: TMEM219 signaling promotes intestinal stem cell death and exacerbates colitis

doi: 10.1172/jci185783

Figure Lengend Snippet: Figure 6. TMEM219 genetic deletion in ISCs ameliorates acute colitis in vivo. (A). Genetic approach used to generate the Tmem219fl/fl EGFP-Lgr5cre mouse, namely the ISC-Tmem219–/– mouse. (B–D). Flow plot and bar graph quantifying TMEM219 protein (B and C) and mRNA (D) expression in EpCam+EGFP- LGR5+ intestinal cells isolated from the Tmem219fl/fl EGFP-Lgr5cre mouse, in which Tmem219 was deleted through tamoxifen injection (ISC-Tmem219–/–, n = 3), compared with the ISC-B6 mice, in which Cre was not activated by tamoxifen injection (n = 3). (E and F). Normalized mRNA expression of Lgr5 and Casp8 in colons of ISC-Tmem219–/– (n = 4) compared with ISC-B6 mice (n = 3). (G). Bar graphs showing ex vivo–generated 8-day miniguts from crypts of ISC- Tmem219–/– (n = 4) and of ISC-B6 controls (n = 5) cultured with/without IGFBP3 (50 ng/mL). (H). Experimental design of the DSS acute prevention model conducted in ISC-Tmem219–/– mice. (I–K). DAI score, colon length, and histological score quantified in ISC-Tmem219–/– mice and in the ISC-B6 control (n = 8–10) with or without treatment with oral DSS (2.5%) in the prevention acute study model described in H. (L). H&E staining of colons obtained from mice as described in I. Arrows highlight inflammation, infiltrating leukocytes. Original magnification ×10; scale bar: 200 μm. (M and N). Flow cytometric analysis of infiltrating CD45+ cells performed in colon samples of ISC-Tmem219–/– mice and of ISC-B6 control mice with or without treatment with oral DSS, (n = 5). (O and P). Ex vivo–generated 8-day miniguts from crypts of ISC-Tmem219–/– mice and of ISC-B6 control control mice with or without treatment with DSS, (n = 6–7). Original magnification ×10; scale bar: 200 μm. Mean ± SEM. 1-way ANOVA followed by Šidák’s post hoc test, 2-sided t test.

Article Snippet: Cleaved Caspase 8 and phosphorylated-AKT were assessed using ELISA (MBS766157, MyBiosource and KHO0111, Invitrogen) in human crypts, patient-derived organoids, and CaCo2 cells cultured with/without IGFBP3 (50 ng/mL, 8874-B3, R&D Systems), with or without ecto-TMEM219 (130 ng/mL, Genscript) (22).

Techniques: In Vivo, Expressing, Isolation, Injection, Ex Vivo, Generated, Cell Culture, Control, Staining