Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: MACC1 activity is directly related to IGFBP2 expression, which interferes with platelet activation. The downregulation of IGFBP2 in the MACC1 KO variant of SW620 cells was indicated at the mRNA level by qPCR ( A ) and confirmed at the protein level by ELISA ( B ). ELISA data confirm the lower expression of IGFBP2 by the MACC1 KO variant of SW620 cells, either in supernatant (s. MACC1 Ctrl vs. s.MACC1 KO, left columns) and at a lower level in the cell lysate (right columns). ( C ) IGF-I is costimulatory for platelet activation and accelerates the effect of 7.5 µM TRAP-6 to induce platelet aggregation. ( D ) IGF-I is associated with platelets and found in platelet plasma or supernatant after activation, but not in considerable amounts in supernatant of SW620 cells. ( E ) Recombinant IGFBP2 is able to block concentration-dependently the activity of TRAP-6 to induce platelet aggregation and ( F ), significantly, the platelet ATP release. *** p < 0.001; **** p < 0.0001.

Article Snippet: The human IGFBP2 and human IGF-I concentrations of the platelet releasates and tumor cell supernatant were determined with human enzyme-linked immunosorbent assays according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: Activity Assay, Expressing, Activation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: IGFBP2 controls platelet activation by SW620 cells. ( A ) The shRNA-mediated knockdown of IGFBP2 in SW620 cells was confirmed by Western blot, compared to nontargeted knockdown IGFBP2 Ctrl, and quantified by IGFBP2 ELISA using cell supernatants (left columns) and cell lysates (right columns) ( B ). ( C ) The knockdown of IGFBP2 in (MACC1-positive) SW620 cells restored the activation potential to platelets in the aggregation assay, or in ATP release ( D ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human IGFBP2 and human IGF-I concentrations of the platelet releasates and tumor cell supernatant were determined with human enzyme-linked immunosorbent assays according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: The impact of IGFBP2 as a functional downstream component of MACC1 on SW620 cell dynamics. ( A,B ) Detection of cell migratory dynamics in a 2D wound healing assay comparing the impact of MACC1 ( A ) and IGFBP2 ( B ) on migration and the effect of platelets to accelerate dynamic properties. ( C ) Analyzing cell invasion in a transmigration assay and the impact of MACC1 knockout or IGFBP2 knockdown. ( D,E ) Analyzing the impact of MACC1 KO or IGFBP2 KD on cell capability to induce coagulation indicates no differences in thrombin formation upon MACC1 KO ( D ) or IGFBP2 KD ( E ) when compared to the respective control cells. ( F ) Flow cytometry data confirm that the knockout of MACC1 in SW620 cells has no impact on tissue factor expression. * p < 0.05; ** p < 0.01, ns = non-significant.

Article Snippet: The human IGFBP2 and human IGF-I concentrations of the platelet releasates and tumor cell supernatant were determined with human enzyme-linked immunosorbent assays according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: Functional Assay, Wound Healing Assay, Migration, Transmigration Assay, Knock-Out, Coagulation, Flow Cytometry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: IGFBP2 is the metastatic factor in vivo. After xenotransplantation of SW620 control or SW620 IGFBP2 KD cells, metastasis formation was inhibited by IGFBP2 knockdown, as shown by ( A ) human satellite DNA and ( B ) immunohistochemistry staining of human cytokeratin 19.

Article Snippet: The human IGFBP2 and human IGF-I concentrations of the platelet releasates and tumor cell supernatant were determined with human enzyme-linked immunosorbent assays according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Immunohistochemistry, Staining

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