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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2019 Apr 26;18(7):1230–1242. doi: 10.1158/1535-7163.MCT-18-0804

Figure 1. Gamma Secretase Inhibition as a single therapeutic agent in prostate cancer cells impairs colony and tumorsphere formation.

Figure 1

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.