Journal: Molecular cancer therapeutics

Article Title: Loss of Notch1 activity inhibits prostate cancer growth and metastasis and sensitizes prostate cancer cells to anti-androgen therapies

doi: 10.1158/1535-7163.MCT-18-0804

Figure Lengend Snippet: A. Immunoblot of human prostate cancer cells: 22RV1, C4–2 and 22RV1 Notch1 knock-out, Delta-Notch1. B. Immunoblot of time-course Gamma Secretase Inhibitors (GSIs) treatment in 22RV1 or C4–2 prostate cancer cells. Cells were treated 24, 48 or 72 hours with Inhibitors DAPT (50 μM) or RO4929097 (20 μM). Staining was performed for activated NICD1 (NICD1 Val1744), Notch1, NICD3, and GAPDH. C. Colony formation assay- 500 cells were plated per well in 6 well dish in triplicate. Cells were grown 9 days, with media and drugs changed every third day. Colonies were then fixed with methanol and stained with 0.1% crystal violet. Colonies were hand counted and graphed as percent colony formation over control treatment. Control treatment (DMSO) was normalized to 100%. Scale bar represents 100mm. Experiment is representative of three, performed in triplicate. D. Tumorsphere formation assay was performed with 1×104 22RV1 or C4–2 cells plated in 50% Matrigel in 24 well plate. Cells were treated with DAPT or RO4929097, then grown for 15 days with media and inhibitors changed every third day. Scale bar represents 250 mm. Error bars are ± SD. Using two-tailed Students t-test: *=P<0.05; ****=P<0.001; ns= no significance.

Article Snippet: B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4.

Techniques: Western Blot, Knock-Out, Staining, Colony Assay, Control, Tube Formation Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Loss of Notch1 activity inhibits prostate cancer growth and metastasis and sensitizes prostate cancer cells to anti-androgen therapies

doi: 10.1158/1535-7163.MCT-18-0804

Figure Lengend Snippet: In vitro assays were performed on 22RV1 and Delta-Notch1 cells. A, B. Colony formation assay- 5×102 cells were plated per well in 6 well dish. Cells were grown 9 days, with media changed every third day. Colonies were then fixed with methanol and stained with 0.1% crystal violet. Colonies were hand counted and graphed as Colony Formation Rate (A) for 22RV1 and Delta-Notch1 cells, or percent colony formation compared to vehicle control (DMSO, normalized to 100%) for treated Delta-Notch1 cells (B). Delta-Notch1 cells were vehicle treated, RO4929097 treated (20 μM) or DAPT treated (50 μM). Scale bars represent 100mm. C, D. Tumorsphere formation assay- 1×104 22RV1 and Delta-Notch1 cells (C), or Delta-Notch1 cells treated with vehicle, RO4929097 (20 μM) or DAPT (50 μM) (D) were plated in 50% Matrigel with RPMI in a 24 well plate. Cells were grown 15 days with media changed every third day. Scale bar represents 250 mm. Experiments performed in triplicate with representative experiments shown. Using two-tailed Students t-test: *=P<0.05; ****=P<0.001; ns= no significance. Error bars are ± SD.

Article Snippet: B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4.

Techniques: In Vitro, Colony Assay, Staining, Control, Tube Formation Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Loss of Notch1 activity inhibits prostate cancer growth and metastasis and sensitizes prostate cancer cells to anti-androgen therapies

doi: 10.1158/1535-7163.MCT-18-0804

Figure Lengend Snippet: For invasion assays, 5×104 cells were plated in Matrigel coated transwell invasion chambers. Cells were drug treated 72 hours prior to serum starving overnight, and plating in chambers. A. 22RV1 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4. B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4. Chambers were incubated 36 hours for 22RV1 and 24 hours for C4–2 cells, then fixed in methanol and stained in 0.1% crystal violet. Five images were captured per chamber at 161X, performed in triplicate chambers and averaged. Experiments were performed concurrently and graphed separately for each cell line for ease of visualization, thus DMSO, DAPT and RO4 conditions are based on the same samples in these graphs. C,D. 2×105 C4–2 cells were resuspended in 20 μl Matrigel and plated as a 3D dot on a 12 well plate. After Matrigel solidified, media was added and wells were treated with ENZ, DAPT, RO4, combined ENZ with DAPT or RO4 (C) or ABI, DAPT, RO4 or combined ABI with DAPT or RO4. Media was changed every 48 hours. Dots were imaged at four leading edges at Day 0 and Day 5 and distance migrated (millimeters mm) was calculated. ENZ and ABI experiments were performed concurrently, then graphed separately for ease of visualization, thus in (C) and (D), DMSO, DAPT and RO4 conditions are based on the same samples. Scale bars = 250μm. All experiments performed in triplicate with representative images shown. *=P<0.05; **=P<0.01; ***=P<0.005; ns= no significance. Error bars +/− SD.

Article Snippet: B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4.

Techniques: Incubation, Staining

Journal: Molecular cancer therapeutics

Article Title: Loss of Notch1 activity inhibits prostate cancer growth and metastasis and sensitizes prostate cancer cells to anti-androgen therapies

doi: 10.1158/1535-7163.MCT-18-0804

Figure Lengend Snippet: A. 22RV1 or C4–2 cells were pre-treated 72 hours with RO4929097 (20 μM) or DAPT (50 μM). Cells were serum starved overnight and 5×104 were plated in Matrigel invasion chambers and incubated 36 or 24 hours respectively. Scale bars = 250μm. B. 22RV1 or Delta-Notch1 cells were serum starved overnight, then 5×104 were plated in Matrigel transwell chambers. Chambers were incubated 36 hours, then fixed with methanol, and stained with crystal violet. For each condition, 5 fields were captured per chamber, number of cells per field were counted, then averaged for three chambers. Error bars represent ±SD. Scale bars = 250 μm C. 1×105 22RV1-Luc or Delta-Notch1-Luc cells were injected intracardiac into NSG mice. Animals were subjected to intraperitoneal (i.p.) injection with 150mg/kg D-Luciferin and imaged at 0 hours, 7 days and 14 days. Final imaging at 14 days is pictured with all animals set to the same radiance scale. D. Quantification of whole animal bioluminescence is plotted as total emission (photons/sec), representing each value in a box and whisker plot on Log10 scale. E. Ex vivo imaging was performed on all organs, with signal maintained in 300μg/ml D-Luciferin after harvest. Instance of organs with positive bioluminescence was graphed out of 6 animals. F. Liver bioluminescence was set to the same radiance scale for all animals, then quantified as radiance (p/sec/cm2/sr) and graphed on Log10 scale in box and whisker plot. G. Representative bioluminescent images of liver from 22RV1-Luc, Delta-Notch1-Luc or negative control (uninjected with cells) animals. Scale bar = 1cm. H. Excised livers were fixed in formalin and paraffin embedded. Tissues were used to perform H&E (5X and 40X) as well as IHC for human KU70. Yellow arrows indicate visible metastatic lesions. Scale bars are 500μm and 50 μm, respectively. I. Representative bioluminescent images of bone metastasis from 22RV1-Luc or Delta-Notch1 conditions. Scale bar = 1cm. *=P<0.05; **=P<0.01; ***=P<0.005

Article Snippet: B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4.

Techniques: Incubation, Staining, Injection, Imaging, Whisker Assay, Ex Vivo, Negative Control

Journal: Molecular cancer therapeutics

Article Title: Loss of Notch1 activity inhibits prostate cancer growth and metastasis and sensitizes prostate cancer cells to anti-androgen therapies

doi: 10.1158/1535-7163.MCT-18-0804

Figure Lengend Snippet: A. Colony formation assay- 5×102 22RV1 or C4–2 cells were plated per well in 6 well dish, treated with vehicle (DMSO) control, Enzalutamide (ENZ) 5 μM; DAPT 50 μM, ENZ + DAPT, or B. DMSO; ENZ; RO4929097 (RO4) 20 μM; or ENZ + RO4. Cells were grown 9 days, with media and drugs changed every third day. Colonies were then fixed with methanol and stained with 0.1% crystal violet. Control treatment (DMSO) was normalized to 100%. C. Colony formation assay was performed for DMSO control, Abiraterone (ABI) 5 μM, DAPT 50 μM, ABI + DAPT, or D. DMSO, ABI, RO4929097 (RO4) 20 μM, or ABI + RO4. Colonies were hand counted and graphed as Percent Colony Formation compared to control (vehicle). Control treatment (DMSO) was normalized to 100%. E. Colony formation of 22RV1 compared against Delta-Notch1 cells in the presence of ENZ, ABI or DMSO control. Graphed as colony formation rate to compare across cell lines. All experiments performed in triplicate with representative images shown. *=P<0.05; ***=P<0.005; ****=P<0.001; ns=no significance. Error bars +/− SD.

Article Snippet: B. C4–2 cells were treated with DAPT (50 μM), ENZ (5 μM), ABI (5 μM), RO4929097 (RO4) (20 μM), or combination of ENZ with DAPT or RO4 and ABI with DAPT or RO4.

Techniques: Colony Assay, Control, Staining