Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: Differentially Expressed ( P < .01) Receptor-Tyrosine Kinase-Associated Genes in Childhood Astrocytomas

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques:

Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: Validation of IGFBP2 differential protein expression in childhood astrocytomas by immunohistochemistry. Representative images demonstrate A) absence of IGFBP2 staining in normal brain, B) weak, diffuse IGFBP2 staining in low-grade astrocytoma, and C) strong, diffuse IGFBP2 staining in high-grade astrocytoma; arrows indicate focal areas of intense IGFBP2 positive staining. Photographed by light microscopy at 200×

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry, Staining, Light Microscopy

Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: Immunohistochemistry Results for IGFBP2 in 13 High-Grade and 18 Low-Grade Childhood Astrocytomas

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques: Immunohistochemistry

Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: IGFBP2 modulation of IGF-mediated astrocytoma cell migration. Transwell migration assay of astrocytoma cells CRL, T98, U87, and U138 on fibronectin stimulated with IGFBP2 alone (50 ng/mL) or IGF-I or IGF-II (10 ng/mL), with or without IGFBP2 (50 ng/mL), for 30 minutes. IGFBP2 minimally inhibited IGF-I-mediated but not IGF-II- mediated cell migration. Increasing concentrations of IGFBP2 alone (5 ng/mL to 50 ng/mL) had no effect on migration over controls (not shown). Optical density (OD) values are the average of three separate experiments and error bars indicate standard deviation. Statistical significance was calculated using a Student t test

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques: Migration, Transwell Migration Assay, Standard Deviation

Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: Differentially Expressed Kinase Genes ( P < .01) in Astrocytoma Cells Following IGFBP2 Stimulation for 1 h and in High-Grade Astrocytomas

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques:

Journal: Journal of child neurology

Article Title: IGFBP2 is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

doi: 10.1177/0883073808321766

Figure Lengend Snippet: IGFBP2 induces DNA-PKcs protein expression in a dose- and time-dependent manner. Western blot of T98 whole cell lysates for DNA-PKcs after 24-hour stimulation with A) IGFBP2 (5 ng/mL to 50 ng/mL), B) IGF-I (1 ng/mL to 10 ng/mL), and C) IGFBP2 (10 ng/mL) measured at 24 hours to 96 hours post-stimulation. Bar graphs show the relative ratio of DNA-PKcs:actin that was used to determine the relative change in DNA-PKcs expression after stimulation with IGFBP-2 and IGF-I. IGF-I had no effect on DNA-PKcs induction over time and similar results were observed with IGFBP-2 stimulation of U87 cells (not shown). Statistical significance was calculated using a Student t test. D indicates DNA-PKcs, A indicates actin control

Article Snippet: Cells were washed to remove endogenous insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) and incubated at 37°C for 30 minutes with recombinant IGF-I (10 ng/mL, R&D Systems) or IGF-II (10 ng/mL, R&D Systems), with or without recombinant IGFBP2 (5 ng/mL to 50 ng/mL, R&D Systems).

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Single cell lineage tracing reveals subclonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

doi: 10.1101/2023.04.04.535588

Figure Lengend Snippet: ( A ) UMAP representation of MDAMB468 cells colour coded respect to the days of treatment. ( B ) Retrospective projection of the afatinib tolerant persisted lineages on UMAP representation of untreated MDAMB468 cells at Day 0. ( C ) Differential expression between afatinib tolerant persisted lineages versus afatinib sensitive lineages on untreated MDAMB468 cells (i.e., cells from Day 0). ( D ) Expression distribution of IGFBP2 in afatinib tolerant persisted lineages and afatinib sensitive lineages across time. Two tailed Wilcoxon test was used for comparison. ( E ) IGFBP2 expression measured by real-time quantitative PCR in parental MDA-MB-468 cell-line (P) versus MDAMB468 afatinib tolerant persisted cells (ATPC). Two tailed t-test was used for comparisons. ( F ) Cellular frequency of afatinib tolerant clones sequenced time points (i.e., CTRL (Day 0), Day 3, Day 6, Day 9). ( G ) Cellular Frequency of clone bc14-013:bc30-092942 and bc14-013:bc30-092942 in the Drug tolerant MDAMB468-ATPC cell line. ( H ) UP-regulated genes comparing cells belonging to the dominant clones versus cells belonging to the neutral clones. ( I ) Average log2 fold change (FC) of the top ten up-regulated genes from (C). 95% confident interval is reported. ( J ) Expression distribution of IGFBP2 in dominant and neutral clones across time. statistical differences were estimated with two-tailed Wilcoxon test.

Article Snippet: The pCMV6-AC-IGFBP2-GFP expression vector encoding human IGFBP2 (NM_000597) fused to the GFP in the C-terminal region was purchased from Origene Technologies (#RG202573).

Techniques: Expressing, Two Tailed Test, Comparison, Real-time Polymerase Chain Reaction, Clone Assay

Journal: bioRxiv

Article Title: Single cell lineage tracing reveals subclonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

doi: 10.1101/2023.04.04.535588

Figure Lengend Snippet: ( A ) Dose-response curve in terms of cell viability following treatment with afatinib at the indicated concentrations on MDAMB468 IGFBP2 knockdown cells and relative control cells. Asterisks indicate that the shift of the two curves is statistically significant. Significance is assessed by using gaussian processes (Methods) and BF is the estimated Bayesian Factor. ( B ) Dose-response curve in terms of cell viability following treatment with afatinib at the indicated concentrations on MDAMB468 IGFBP2 overexpressing cells and relative control cells. ( C ) Colony assay (representative image) after 10 days of afatinib exposure at 2, 5 and 10μM for MDAMB468 IGFBP2 overexpressing cells and relative control cells (left). Quantification is reported on the right. Experiments were performed in triplicate. ( D ) Spheroid volume growth with 2μM of afatinib (see methods) over time of MDAMB468 IGFBP2 overexpressing cells and relative control cells. ( E ) Colony assay (representative image) after 3 days of afatinib exposure at 0.5 and 1μM for MDAMB468 IGFBP2 knockdown cells and relative control cells (left). Quantification is reported on the right. Experiments were performed in triplicate. ( F ) Spheroid volume growth with 2μM of afatinib over time of MDAMB468 IGFBP2 knockdown cells and relative control cells. ( G ) Spheroid volume growth over time of MDAMB468 IGFBP2 overexpressing cells. ( H ) Spheroid volume growth over time of MDAMB468 IGFBP2 overexpressing cells. ( I ) Transwell migration assay of MDAMB468 IGFBP2 overexpressing cells. Reported p values from panels C to I are estimated using two-tailed t-test.

Article Snippet: The pCMV6-AC-IGFBP2-GFP expression vector encoding human IGFBP2 (NM_000597) fused to the GFP in the C-terminal region was purchased from Origene Technologies (#RG202573).

Techniques: Knockdown, Control, Colony Assay, Transwell Migration Assay, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on PD‐like phenotypes induced by 6‐OHDA in rats. (A) Schematic representation showing the experiment process of this study. (B, C) RT‐qPCR and western blotting analysis revealed that IGFBP2 expression was downregulated in the substantia nigra pars compacta (SNpc) of 6‐OHDA‐induced PD rats. (D) Behavioral tests showing the effect of IGFBP2 on 6‐OHDA‐induced behavioral impairment (Rotation, F (2, 15) = 745.7; Latency time, F (2, 15) = 59.48; T ‐turn time, F (2, 15) = 33.01; T ‐total time, F (2, 15) = 95.94). (E) Representative images showed IHC staining of TH in the SNpc of 6‐OHDA‐lesioned rats, followed by quantification of the number of TH‐positive cells ( F (2, 15) = 188.2). (F) Western blotting analysis depicting α‐synuclein expression in the SNpc tissues ( F (2, 15) = 102.3). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Differential expression of mRNAs in PD rats. (A) The rats were injected with 6‐OHDA to induce the PD animal model. Apomorphine‐induced rotational behavior was tested at 2 and 3 weeks after modeling. (B) IHC staining of TH in OHDA‐lesioned rats (3 weeks). Bar = 100 µm. (C) Bidimensional principal component analysis (PCA) showing distinct clustering of gene profiles in 6‐OHDA‐induced PD rats and control rats. (D, E) Volcano plot and heatmap showing differential expression of mRNAs (|log 2 fold change (FC)| > 1 and p < 0.05). (F) Venn graph depicting the common genes of downregulated differentially expressed genes (DEGs, log 2 FC < −1.5 and p < 0.001) and parkinson‐related genes from the GeneCards database. (G) The FPKM value of IGFBP2 was shown. Group sizes were: N = 3 animals per group. ** p < 0.01, *** p < 0.001. * = control vs. 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Injection, Animal Model, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on 6‐OHDA‐induced oxidative stress and mitochondrial impairment in rats. (A) ROS production, MDA content, and SOD and GSH‐Px activities in the SNpc were tested using corresponding commercial assay kits (ROS, F (2, 15) = 37; MDA, F (2, 15) = 42.56; SOD, F (2, 15) = 25.42; GSH‐Px, F (2, 15) = 45.15). (B) JC‐1 staining indicating the changes in mitochondrial membrane potential (Ψm; Q2‐2, F (2, 15) = 175.4; Q2‐4, F (2, 15) = 175.5). (C) ATP level was assessed by ATP detection kit ( F (2, 15) = 26.63). (D) Western blotting analysis revealing cytochrome c expression in cytoplasm and mitochondria in the SNpc of rats (cytochrome c in cytoplasm, F (2, 15) = 38.49; cytochrome c in mitochondria, F (2, 15) = 193.7). (E) Representative images of TUNEL‐positive staining in the SNpc tissues. Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Membrane, Western Blot, Expressing, TUNEL Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on IGF‐1R/AKT signaling pathway. (A, B) Relative expression levels of IGF‐1R, p‐IGF‐1R, AKT, and p‐AKT in the SNpc were measured by western blotting (p‐IGF‐1R, F (2, 15) = 345.4; IGF‐1R, F (2, 15) = 229.7; p‐IGF‐1R/IGF‐1R, F (2, 15) = 92.01; p‐AKT, F (2, 15) = 811.5; AKT, F (2, 15) = 0.2335; p‐AKT/AKT, F (2, 15) = 851.2). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on apoptosis induced by 6‐OHDA in differentiated PC12 cells. The differentiated PC12 cells were co‐treated with rIGFBP2 and rIGF‐1 for 24 h, and then subjected to 6‐OHDA for 24 h. (A) CCK‐8 assay was performed to determine cell viability ( F (4, 10) = 45.19). (B) LDH content was evaluated using a LDH detection kit ( F (4, 10) = 45.50). (C) Apoptosis induced by 6‐OHDA was determined with Hoechst 33258 staining ( F (4, 10) = 160.7). (D) Bax, Bcl‐2, cleaved caspase‐9, and cleaved caspase‐3 expression were determined by western blotting in PC12 cells (Bax, F (4, 10) = 82.57; Bcl‐2, F (4, 10) = 136.6; cleaved caspase‐9, F (4, 10) = 53.36; cleaved caspase‐3, F (4, 10) = 65.57). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. * p < 0.05, *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001, + p < 0.05, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: CCK-8 Assay, Staining, Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on oxidative stress and mitochondrial function in 6‐OHDA‐lesioned PC12 cells. (A) ROS and MDA levels, SOD and GSH‐Px activities were determined by the commercial assay kits (ROS, F (4, 10) = 37.55; MDA, F (4, 10) = 41.91; SOD, F (4, 10) = 55.79; GSH‐Px, F (4, 10) = 75.03). (B) Changes in Ψm were evaluated by JC‐1 staining. (C) Relative expression of Cytochrome c in cytoplasm and mitochondria was detected by western blotting analysis (cytochrome c in cytoplasm, F (4, 10) = 47.39; cytochrome c in mitochondria, F (4, 10) = 62). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ## p < 0.01, ### p < 0.001, ++ p < 0.01, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Expressing, Western Blot, Control