Journal:

Article Title: Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism

doi: 10.1096/fj.08-118976

Figure Lengend Snippet: IGF-binding proteins and IGF-1 clearance. A) Half-life of [125I]IGF-1 in sera obtained from control, LID, ALSKO, and BP3KO mice. Data are means ± se; n >12 mice/group. *P < 0.05 vs. control. B) Ligand blot assay shows IGF-1 binding capacity of serum from control, LID, ALSKO, BP3KO, and LAB mice. Reactive bands corresponding to IGFBP-3 were not detectable in BP3KO and LAB mice and significantly reduced in LID and ALSKO mice. C) Serum IGFBP-2 levels, determined by RIA in sera from 8- to 10-wk-old control, LID, ALSKO, BP3KO, and LAB mice. Data are means ± se (number per group in parentheses). D) Serum levels of IGFBP-5 detected by Western immunoblot and (E) quantified by densitometry, showing twofold increase in BP3KO mice. Data are mean ± se; n = 5/group.

Article Snippet: Serum levels of mouse IGFBP-2, IGFBP-3, and ALS were determined using ELISA assays developed at UCLA, with recombinant mouse proteins from R&D Systems (Minneapolis, MN, USA) and monoclonal antibodies as described previously (29) .

Techniques: Binding Assay, Western Blot

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: Quantitative Proteomics, 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