Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 up-regulation and increased secretion into culture media by primary and immortalized human esophageal cells transduced with EGFR. A. IGFBP-3 mRNA was determined by real-time RT-PCR in primary human esophageal cells and their derivatives. Mean ± SE (n = 3) in a representative experiment is shown. B. Protein expression for EGFR and IGFBP-3 was determined in cell lysates by Western blotting. β-Actin was used as a loading control. Lane 1, parental EPC2; Lane 2, EPC2-GFP; Lane 3, EPC2-EGFR; Lane 4, EPC2-Neo; Lane 5, EPC2-EGFR; Lane 6, EPC2-hTERT; Lane 7, EPC2-hTERT-Neo; Lane 8, EPC2-hTERT-EGFR; Lane 9, EPC1-Neo; Lane 10, EPC1-EGFR. C. IGFBP-3 secreted by cells into CM was determined by Western blotting using concentrated (10×) CM. D. IGFBP-3 concentration in CM from EPC1, EPC2, and their derivatives was measured by ELISA. For protein analysis, 1 × 106 cells were seeded per 100-mm plate, and medium exchange was done 48 hours before harvesting the CM. Protein yields in cell lysates prepared simultaneously were used to adjust the differences in cell number that may affect IGFBP-3 concentration in CM. Lane 1, EPC2-Neo; Lane 2, EPC2-EGFR; Lane 3, EPC2-hTERT; Lane 4, EPC2-hTERT-Neo; Lane 5, EPC2-hTERT-EGFR; Lane 6, EPC1-Neo; Lane 7, EPC1-EGFR. E and F, immunofluorescence for IGFBP-3 in EPC2-hTERT-EGFR (E) and EPC2-hTERT-neo (F) cells. Magnification, ×400. PD, population doublings.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 is up-regulated in primary esophageal cells transduced with EGFR in organotypic culture and EGFR transgenic mouse esophagus. IGFBP-3 immunohistochemistry in the reconstituted epithelium of (A) EPC2-EGFR and (B) EPC2-Neo cells in organotypic culture. Note that IGFBP-3 was also detected in quiescent fibroblasts embedded in the collagen matrix (Col) underlying the reconstituted epithelia (Epi). Magnification: A and B, ×200. C. IGFBP-3 mRNA level was measured by real-time RT-PCR in the liver and esophagus of an EGFR transgenic mouse and control wild-type mouse. Mean ± SE (n = 3) in a representative experiment is shown. D. Esophageal epithelial cells (mouse esophageal keratinocytes) isolated from the EGFR transgenic mouse (MEK-EGFR) and the control mouse (MEK-WT) were grown in culture and subjected to real-time RT-PCR for IGFBP-3 mRNA. Mean ± SE (n = 3) in a representative experiment is shown. E. Western blotting was carried out to determine EGFR and IGFBP-3 protein expressed in cell lysates and CM, respectively. Tubulin was used as a loading control. *, P < 0.01.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Transduction, Transgenic Assay, Immunohistochemistry, Quantitative RT-PCR, Isolation, Western Blot

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 is overexpressed in primary esophageal cancer tissues. IGFBP-3 mRNA was determined by real-time RT-PCR in paired tissues of tumor and adjacent normal mucosa. Nineteen ESCC (A) and seven EAC (B) samples were analyzed. Mean ± SE (n = 3) in a representative experiment is shown. IGFBP-3 overexpression was based on ≥3-fold IGFBP-3 mRNA levels in tumor specimens (■) compared with normal tissue specimens (□). C. EGFR and IGFBP-3 were determined in tissue lysates prepared from 11 paired clinical samples by Western blotting. T, tumor; N, adjacent normal tissue. Tubulin was used as a loading control.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Quantitative RT-PCR, Over Expression, Western Blot

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 protein is highly expressed in primary esophageal cancer. IGFBP-3 immunohistochemistry is shown in tissue sections of (A) normal esophageal epithelium, (B) squamous dysplasia, (C–E) invasive ESCC, and (F–H) EAC. A–D represent clinical specimen ESCC4; F and G represent EAC1 in which IGFBP-3 mRNA levels were determined, and H represents EAC6 in which IGFBP-3 mRNA levels were determined (Fig. 3). Intensity was + in A, ++ in B and F, and +++ in C–E, G, and H. The insets in C and G were shown with higher magnification as D and H, respectively. Magnification: C, E, and G, ×100; A, B, and F, ×200; D and H, ×400.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Immunohistochemistry

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 is overexpressed in esophageal cancer cell lines. A. IGFBP-3 mRNA was determined by real-time RT-PCR in primary human esophageal cells EPC1 and EPC2; EAC cell line TE7; ESCC cell lines TE1, TE2, TE3, TE5, TE6, TE8, TE9, TE10, TE11, TE12, TE15, T.T, HCE4, and HCE7; and nonesophageal cell lines. Mean ± SE (n = 3) in a representative experiment is shown. B. IGFBP-3 expression was determined by Western blotting using cell lysates from the indicated cell lines. Tubulin was used as a loading control. C. IGFBP-3 secreted by cell lines into DMEM-0.5% FCS was determined by Western blotting using concentrated (10×) CM. D. ELISA was done using CM without concentration. For protein analysis shown in C and D, 1 × 106 cells were seeded per 100-mm plate. Medium change was done 48 hours before harvesting CM with either full medium (DMEM-10% FCS) or starving medium (DMEM-0.5% FCS). Protein yield in cell lysates prepared simultaneously was used to adjust the difference in cell number that may affect IGFBP-3 concentration in CM.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

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

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 secreted by cancer cell lines is capable of binding to IGF-I. A. 10×-concentrated CM was examined for 125I -labeled IGF-I binding activity by Western ligand blotting. A nonspecific band (*), present not only in CM from the indicated cell lines but also in 0.5% FCS, is likely bovine IGFBP. The identical blot was reprobed with antibodies specific for IGFBP-4 (B) and IGFBP-3 (C) to identify the IGF-I binding activities shown in A.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Binding Assay, Labeling, Activity Assay, Western Blot

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: IGFBP-3 expression is regulated by EGFR activation. IGFBP-3 expression in cell lysates and CM was determined by ELISA (A, C, E, and G) and Western blotting (B, D, and F) after treatment with AG1478 (specific EGFR tyrosine kinase inhibitor), dimethyl sulfoxide as a control vehicle, or EGF at the indicated concentrations and for the indicated periods. A. TE2, TE7, and TE11 cells were incubated with or without AG1478 for 72 hours in the presence of 10% FCS, and CM was subjected to ELISA. B–G. TE11 (B and C), TE2 (D and E), and TE7 (F and G) cells were serum starved with serum-free medium for 16 hours and treated with or without EGF for 48 hours. *, P < 0.01.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: Cancer research

Article Title: Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

doi: 10.1158/0008-5472.CAN-04-0715

Figure Lengend Snippet: Cell growth is enhanced in TE11 cells by siRNA-mediated suppression of IGFBP-3. Expression of IGFBP-3 in TE11 cells stably transduced with siRNA against IGFBP-3 (TE11-S) and control cells (TE11-N) was determined by Western blotting (A) and ELISA (B). A total of 1 × 106 cells were seeded per 100-mm plate, and medium was exchanged 48 hours before harvesting cell lysates and CM. β-Actin was used as a loading control for Western blotting. Protein yields in cell lysates were used to adjust the differences in cell number that may affect IGFBP-3 concentration in CM. C. Growth curves were drawn by seeding cells in 6-well plates (5.0 × 104 cells per well) in a triplicate fashion and counting them at the indicated time points. D. DNA synthesis was measured in subconfluent TE11-S and TE11-N cells. Mean ± SE (n = 4) represents one of two independently performed experiments with similar results. *, P < 0.01.

Article Snippet: After blocking with 1% bovine serum albumin (Sigma) for 10 minutes, slides were incubated with antihuman IGFBP-3 mouse monoclonal antibody (clone 84728.111; R&D Systems Inc.) at a 1:250 dilution overnight at 4°C and then incubated with Cy3-conjugated secondary donkey antimouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution for 30 minutes at 37°C.

Techniques: Expressing, Stable Transfection, Transduction, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, DNA Synthesis

Journal: Aging and Disease

Article Title: Reduction in IGF1 mRNA in the Human Subependymal Zone During Aging

doi: 10.14336/AD.2018.0317

Figure Lengend Snippet: Pearson’s product-moment correlations between gene expression of IGF family members and brain cohort characteristics in the human SEZ.

Article Snippet: mRNA levels were measured by TaqMan Gene Expression Assays (Applied Biosystems; IGF1, Hs01547656_m1; IGF1R, Hs00609566_m1; IGFBP2, Hs01040719_m1; IGFBP3, Hs00181211_m1; IGFBP4, Hs01057900_m1; IGFBP5, Hs00181213_m1; INSR, Hs00961557_m1) using an ABI Prism 7900HT fast real-time PCR system and a 384-well format.

Techniques: Gene Expression

Journal: eNeuro

Article Title: Neurochemical Heterogeneity Among Lateral Hypothalamic Hypocretin/Orexin and Melanin-Concentrating Hormone Neurons Identified Through Single-Cell Gene Expression Analysis

doi: 10.1523/ENEURO.0013-17.2017

Figure Lengend Snippet: Panel of 48 genes used to probe the neurochemical profile of Hcrt/Ox and MCH neurons

Article Snippet: Insulin-like growth factor binding protein 3 (IGFBP3) , Igfbp3 , Growth factor binding , Mm01187817_m1.

Techniques: Sequencing, Marker, Binding Assay, RNA Binding Assay

Journal: eNeuro

Article Title: Neurochemical Heterogeneity Among Lateral Hypothalamic Hypocretin/Orexin and Melanin-Concentrating Hormone Neurons Identified Through Single-Cell Gene Expression Analysis

doi: 10.1523/ENEURO.0013-17.2017

Figure Lengend Snippet: Proportional difference in expression of all 48 genes in Hcrt/Ox and MCH neurons

Article Snippet: Insulin-like growth factor binding protein 3 (IGFBP3) , Igfbp3 , Growth factor binding , Mm01187817_m1.

Techniques: Expressing

Journal: Communications Biology

Article Title: Mucosal ribosomal stress-induced PRDM1 promotes chemoresistance via stemness regulation

doi: 10.1038/s42003-021-02078-1

Figure Lengend Snippet: a The intestinal cancer cell lines (the negative control plasmid- or shIGFBP3-expressing HCT-8) were treated with DMSO, ID 90 of RIS-1 or ID 90 of RIS-3. Total cell lysates were subjected to western blot analysis. Figures in the lower box indicate IGFBP3 mRNA expression in cell lines. b Mouse tissues from allograft CRC were analyzed using immunohistochemistry for IGFBP3, and pERK1/2 (left panels) with hematoxylin counterstaining (magnification ×400; scale bar(s), 100 μm). Quantification of DAB staining from IHC analysis (right graphs). Results are shown as a plot with Tukey whiskers and asterisks represent a significant difference compared to the control group ( n = 11 or 14, *** P < 0.001). c p53 wildtype (+/+) and mutant (−/−) HCT-116 cells were treated with DMSO or 5-FU for 48 h. Total cell lysates were subjected to western blot analysis. d The intestinal cancer cells (the control vector-, shPRDM1- or PRDM1 overexpression plasmid-transfected HCT-8 cells) were treated with DMSO or 375 μM 5-FU for 48 h. Total cell lysates were subjected to western blot analysis. e The intestinal cancer cell lines (the control plasmid-, shPRDM1- or shIGFBP3-expressing HCT-8) pre-exposed to the vehicle, ID 80 of RIS-1 or 50 ng/ml IGFBP3 for 24 h were treated with 375 μM 5-FU for 48 h and mRNA levels were measured using RT real-time PCR. Results are shown as mean values ± SD and different letters (a–f) over each bar represent significant differences between groups ( n = 4–8, P < 0.05).

Article Snippet: Proteins were transferred onto PVDF membrane (Pall Corporation, New York, NY, USA), after which the blots were blocked for 1 h with 5% skimmed milk in Tris-buffered saline plus Tween 0.1% (TBST) and probed with a 1:1000 dilution of each primary antibody (rabbit polyclonal anti-β-actin, mouse monoclonal anti-p53, goat polyclonal anti-Gdf15, rabbit polyclonal anti-RhoA, mouse monoclonal anti-PRDM1, mouse monoclonal anti-pERK1/2, mouse monoclonal anti-pJNK1/2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-pGSK3b (S9), rabbit polyclonal anti-CDX2 (ABclonal, Wuhan, China), or rabbit polyclonal anti-IGFBP3 (Bioss Antibodies, Woburn, MA, USA)) for additional 2 h at room temperature or overnight at 4 °C.

Techniques: Negative Control, Plasmid Preparation, Expressing, Western Blot, Immunohistochemistry, Staining, Mutagenesis, Over Expression, Transfection, Real-time Polymerase Chain Reaction