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: 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