Journal: Cancer cell

Article Title: Overcoming Resistance to Dual Innate Immune and MEK Inhibition Downstream of KRAS

doi: 10.1016/j.ccell.2018.08.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-phospho IGF1R β (Tyr1135) , Cell signaling technology , Cat# 3918S; RRID: AB_10548764.

Techniques: Control, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Viability Assay, shRNA, Negative Control, Software, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice

doi: 10.1371/journal.pone.0224674

Figure Lengend Snippet: A, B, Immunoblots (A) and quantitative densitometry (B) showing the levels of p-Creb, G6Pase and PEPCK1 in the livers of NC-fed mice and HFD-fed mice treated with vehicle (Veh) or carvedilol (Carv). Normalized to total Creb or GAPDH. C, D, PTT (C) and PTT AUC (D) of NC-fed mice and HFD-fed mice treated with vehicle or carvedilol. E, Plasma insulin of NC-fed mice and HFD-fed mice treated with carvedilol or vehicle. F, G, Immunoblots (F) and quantitative densitometry (G) showing the levels of p-Creb, p-InsR and p-Akt in the muscles of NC-fed mice and HFD-fed mice treated with vehicle (Veh) or carvedilol (Carv). Normalized to total Creb, Akt or GAPDH. Data are presented as mean ± S.E.M. One-way ANOVA with Turkey’s post-tests in bar graphs and two-way ANOVA with Bonferroni’s post-tests in line graphs. *P< 0.05, **P<0.01 compared to NC group, #P<0.05 compared to HFD+Veh group. Hormone measurement and Western blot analysis were performed in triplicate.

Article Snippet: Primary antibodies including Akt (Cat. No. 2920, dilution 1:10,000), p-Akt (Cat. No. 3787, dilution 1:10,000), Creb (Cat. No. 9197, dilution 1:2,000), p-Creb (Cat. No. 9196, dilution 1:2,000), PEPCK1 (Cat. No. 12940, dilution 1:2,000) and p-IGF-1Rβ/ InsRβ (Cat. No. 3021, dilution 1:2,000) were obtained from Cell Signaling (Cell Signaling Technology Inc., MA, USA).

Techniques: Western Blot, Clinical Proteomics, Muscles

Journal: bioRxiv

Article Title: Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

doi: 10.1101/253278

Figure Lengend Snippet: (A) SUM44PE, (B) MDA-MB-134, and (C) BCK4 ILC cells were immunostained for IGF1R (green) and E-cadherin (red) and imaged by confocal microscopy. Of note, BCK4 cells were imaged at an increased exposure compared to MM134 and SUM44PE cells. (D) CDH1 mRNA, (E) IGF1 mRNA, (F) and plGF1 R Y1135 & Y1136 levels in ER+ IDC compared to ER+ ILC in TCGA were plotted using RNAseq (Iog2 TPM+1) and RPPA (median normalized) data. The TCGA cohort includes n=417 IDC cases and n=137 ILC cases that have matched data for RNAseq and RPPA. Man-Whitney test was used to determine significant differences in expression level between the two subtypes, p<0.05). (G) Correlation between pIGFIR and IGF1 ligand expression is plotted for IDC (left) and ILC (right). Spearman’s rank correlation was used to demonstrate the correlation between the two variables with significance as defined by p<0.05.

Article Snippet: Membranes were blocked in Odyssey PBS Blocking Buffer (LiCor #927-40000), and incubated in primary antibodies overnight: plGF1RY1135 (Cell Signaling #3918; 1:500), IGF1R β-subunit (Cell I Signaling #3027; 1:1000), pAkt S473 (Cell Signaling #4060; 1:1000), total Akt (Cell Signaling i #9272; 1:1000), E-cadherin (BD Biosciences #610182; 1:1000), and β-actin (Sigma #A5441; 1:5000).

Techniques: Confocal Microscopy, Expressing

Journal: bioRxiv

Article Title: Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

doi: 10.1101/253278

Figure Lengend Snippet: (A) MCF-7, (B) ZR75.1, and (C) T47D breast cancer cells transfected with SCR (siSCR) or CDH1 (siCDHI) siRNA were stimulated with increasing doses of IGF1 (0-100nM) for 10 min. IGF1R and Akt signaling was assessed by immunoblot. Of note, IGF1R expression could routinely not be detected in ZR75.1. (D) MCF-7 cells were treated with 25ug/ml HECD-1 antibody for 24 hours and imaged by phase-contrast microscopy for dissociation of adherens junctions. Cells were stimulated with Vhc or 10nM IGF1 for 10 min and IGF1R and Akt signaling assessed by immunoblot. (E) MCF-7 cells were plated at sub-confluency (200k cells in 6-well) or high confluency (800k cells) and then stimulated with either Vhc or 10nM IGF1 for 10 min. IGF1R signaling was assessed by immunoblot. Representative phase-contrast microscopy images of the cell plating densities are shown. (F) MCF-7 and ZR75.1 siSCR and siCDHI cells were serum-starved and stimulated with 10nM IGF1 for 17 hours and DNA stained with propidium iodide to measure cell cycle profile. The percent of cells in the IGF1 A/hc conditions in the S- and G2/M phases of the cell cycle for siSCR and siCDHI are shown (representative experiment shown; n=2 or 3 each with 3 biological replicates).

Article Snippet: Membranes were blocked in Odyssey PBS Blocking Buffer (LiCor #927-40000), and incubated in primary antibodies overnight: plGF1RY1135 (Cell Signaling #3918; 1:500), IGF1R β-subunit (Cell I Signaling #3027; 1:1000), pAkt S473 (Cell Signaling #4060; 1:1000), total Akt (Cell Signaling i #9272; 1:1000), E-cadherin (BD Biosciences #610182; 1:1000), and β-actin (Sigma #A5441; 1:5000).

Techniques: Transfection, Western Blot, Expressing, Microscopy, Staining

Journal: bioRxiv

Article Title: Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

doi: 10.1101/253278

Figure Lengend Snippet: MCF-7 cells were reverse transfected with SCR or CDH1 siRNA and seeded into 96-well 2D or ULA plates and treated with IGF1R inhibitor (OSI-906 or BMS-754807) for 6 days. Conditions in the panels as follows: (A) OSI-906; 2D, (B) OSI-906; ULA, (C) BMS-754807; 2D, (D) BMS-754807; ULA. The CellTiter Glo assay was used to assess cell viability (relative luminescence). EC50 values for viability were calculated by non-linear regression and statistical differences evaluated using sum-of-squares Global f-test (p<0.05; representative experiment shown; n=3 each with 6 biological replicates).

Article Snippet: Membranes were blocked in Odyssey PBS Blocking Buffer (LiCor #927-40000), and incubated in primary antibodies overnight: plGF1RY1135 (Cell Signaling #3918; 1:500), IGF1R β-subunit (Cell I Signaling #3027; 1:1000), pAkt S473 (Cell Signaling #4060; 1:1000), total Akt (Cell Signaling i #9272; 1:1000), E-cadherin (BD Biosciences #610182; 1:1000), and β-actin (Sigma #A5441; 1:5000).

Techniques: Transfection, Glo Assay

Journal: bioRxiv

Article Title: Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

doi: 10.1101/253278

Figure Lengend Snippet: In situ proximity ligation assay (PLA) was used to analyze the direct interaction between IGF1R and E-cadherin in breast cancer cells. (A) MCF-7 and (B) T47D cells were plated on coverslips, fixed, and stained with IGF1R and E-cadherin antibody overnight. The Duolink (Sigma) protocol was followed and coverslips were imaged using confocal microscopy to reveal red puncta. (C) MCF-7 siCDHI and (D) silGF1R cells were used as negative controls for the assay to assess primary antibody specificity. MCF-7 cells were plated on coverslips and treated with either (E) Vhc or 10nM IGF1 for (F) 30 minutes, (G) 6 hours, or (H) 24 hours. PLA protocol for IGF1R and E-cadherin was followed as described above. (I) Red puncta and nuclei (stained with DAPI) were quantified and displayed as a ratio of puncta/nuclei. All puncta and nuclei in 60x images were counted. Oneway ANOVA was used to determine significant difference between groups (p<0.05; one independent experiment, n=5 images per slide counted). The co-localization of IGF1R (green) and E-cadherin (red) was analyzed by immunofluorescence staining in (J) MCF-7 siSCR and (K) siCDHI knockdown cells.

Article Snippet: Membranes were blocked in Odyssey PBS Blocking Buffer (LiCor #927-40000), and incubated in primary antibodies overnight: plGF1RY1135 (Cell Signaling #3918; 1:500), IGF1R β-subunit (Cell I Signaling #3027; 1:1000), pAkt S473 (Cell Signaling #4060; 1:1000), total Akt (Cell Signaling i #9272; 1:1000), E-cadherin (BD Biosciences #610182; 1:1000), and β-actin (Sigma #A5441; 1:5000).

Techniques: In Situ, Proximity Ligation Assay, Staining, Confocal Microscopy, Immunofluorescence

Journal:

Article Title: IGFBP-3, a Marker of Cellular Senescence, Is Overexpressed in Human Papillomavirus-Immortalized Cervical Cells and Enhances IGF-1-Induced Mitogenesis

doi: 10.1128/JVI.78.11.5720-5727.2004

Figure Lengend Snippet: IGFBP-3 augments ligand activation of the IGF-1R. (A) Dose response of IGFBP-3 effects on IGF-1R activation. Cultured cervical cells were maintained for 3 days in basal medium containing various concentrations of IGFBP-3. The cells were then treated with 25 ng of IGF-1/ml for 10 min, lysed, and analyzed for tyrosine phosphorylation of the IGF-1R by immunoblotting as described in the text. The arrow indicates the position of the IGF-1R β-subunit. (B) Effects of immediate and prolonged IGFBP-3 exposure on IGF-1R activation. Early-passage cervical cells were exposed to IGFBP-3 at the same time as they received IGF-1 for 0 days or, alternatively, they were exposed to IGFBP-3 for 3 days prior to addition of IGF-1 (3 days). Late-passage cells responded to exogenous IGF-1 with enhanced IGF-1R phosphorylation. In early-passage cells, a similar increase in tyrosine-phosphorylated (pY) IGF-1R was observed after preincubation with IGFBP-3, indicating their conversion to the IGF-1-responsive phenotype of late-passage cells. Simultaneous IGFBP-3-IGF-1 incubation of early-passage cells diminished this effect, suggesting a necessity for preincubation with IGFBP-3.

Article Snippet: A 50-μg aliquot of total protein was separated in an 8% Tris-glycine gel (Invitrogen), transferred to an Immobilon-P membrane (Millipore), and labeled for 90 min with 0.2 μg of anti-IGF-1Rβ rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)/ml diluted in washing buffer (0.5% Triton X-100, 140 mM NaCl, 10 mM Na 3 PO 4 ) in the presence of 2% bovine serum albumin (ICN Biomedicals, Inc., Aurora, Ohio).

Techniques: Activation Assay, Cell Culture, Phospho-proteomics, Western Blot, Incubation

Journal:

Article Title: IGFBP-3, a Marker of Cellular Senescence, Is Overexpressed in Human Papillomavirus-Immortalized Cervical Cells and Enhances IGF-1-Induced Mitogenesis

doi: 10.1128/JVI.78.11.5720-5727.2004

Figure Lengend Snippet: Total and cell surface IGF-1R levels remain stable during cell passaging. (A) Detection of total cell IGF-1R. Total IGF-1R protein levels were detected by Western blotting at the indicated passages using an antibody that specifically binds to the IGF-1R β-chain. NWT21 cells (a mouse 3T3 fibroblast cell line overexpressing the IGF-1R) served as a positive control. The positions of the IGF-1R β-subunit and the IGF-1R precursor are indicated. (B) Detection of cell surface IGF-1R. Cell surface IGF-1R levels were evaluated on live cervical cells by flow cytometry using the indicated antibody. The number of cells versus relative fluorescence intensity is plotted. The levels of total and surface IGF-1R showed no significant changes during cell passaging.

Article Snippet: A 50-μg aliquot of total protein was separated in an 8% Tris-glycine gel (Invitrogen), transferred to an Immobilon-P membrane (Millipore), and labeled for 90 min with 0.2 μg of anti-IGF-1Rβ rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)/ml diluted in washing buffer (0.5% Triton X-100, 140 mM NaCl, 10 mM Na 3 PO 4 ) in the presence of 2% bovine serum albumin (ICN Biomedicals, Inc., Aurora, Ohio).

Techniques: Passaging, Western Blot, Positive Control, Flow Cytometry, Fluorescence

Journal:

Article Title: IGFBP-3, a Marker of Cellular Senescence, Is Overexpressed in Human Papillomavirus-Immortalized Cervical Cells and Enhances IGF-1-Induced Mitogenesis

doi: 10.1128/JVI.78.11.5720-5727.2004

Figure Lengend Snippet: IGFBP-3 enhances IGF-1R downstream signaling. Cells were incubated for 3 days in basal medium containing various concentrations of IGFBP-3. The cultures were then treated with IGF-1 for 10 min, and cell lysates were analyzed by immunoprecipitation and immunoblotting for the amount of tyrosine-phosphorylated (pY) IRS. After detection of (pY) IRS levels, the same membrane was stripped and reprobed for total IRS levels as described in the text.

Article Snippet: A 50-μg aliquot of total protein was separated in an 8% Tris-glycine gel (Invitrogen), transferred to an Immobilon-P membrane (Millipore), and labeled for 90 min with 0.2 μg of anti-IGF-1Rβ rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)/ml diluted in washing buffer (0.5% Triton X-100, 140 mM NaCl, 10 mM Na 3 PO 4 ) in the presence of 2% bovine serum albumin (ICN Biomedicals, Inc., Aurora, Ohio).

Techniques: Incubation, Immunoprecipitation, Western Blot, Membrane