Supplemental Figure 3. PF-03084014 Inhibits Notch Pathway in Prostate Cancer Tumor Xenografts

Supplemental Figure 1. Genetic knockdown of Notch-1 inhibits growth and docetaxel chemoresistance of prostate cancer cells similar to PF-03084014. (A-C) PC3R or Du145R cells were transiently transduced with either Notch-1 miRNA or scrambled (SCR) control vectors. (A) Cells were subjected to Western using β-actin was used as loading control. Bands were measured using densitometry and values first normalized to respective β-actin bands and then reported below each gel as relative to each cells scrambled control. (B) 2.5×103 prostate cancer cells were plated per well in 96-well plates and allowed to grow for 48 hours. Cell viability was measured using resazurin cell viability assays. Results are reported as optical density (O.D.). (C) 2.5×103 prostate cancer cells were plated per well in 96-well plates and treated with either vehicle (DMSO) or docetaxel (10μM) for 48 hours. Cell viability was measured using resazurin cell viability assays. Results are reported as the percentage of live cells in docetaxel-treated group compared to the DMSO vehicle-treated group. (E-G) PC3R or Du145R cells were transiently transduced with either NICD or empty vector control plasmids. (A) Cells were subjected * p<0.05 versus vector.

Supplemental Figure 2. PF-03084014 combined with docetaxel suppresses tumor cell proliferation, induces apoptosis and inhibits tumor angiogenesis in mice.

At end of study, tumors were harvested from the mice described in Figure 2. A portion of the Du145R tumor xenografts (n=5) were subjected to immunohistochemistry for proliferation (anti-Ki67), apoptosis (anti-cleaved Caspase 3) and tumor angiogenesis (anti-CD31). Left: Representative photomicrographs of Ki67, cleaved-caspase 3 and CD31 stained tumor sections (×40). Right: Quantitation of Ki-67, cleaved-caspase 3 and CD31 positive percentage (* p<0.05).

Supplemental Figure 3. PF-03084014 inhibits notch pathway in prostate cancer tumor xenografts in vivo.

At end of study, tumors were harvested from the mice described in Figure 2. Protein and mRNA were extracted from a portion of the DU145 and Du145R tumor xenografts. (A) Proteins were subjected to immunoblot analysis for NICD. β-actinwas used as loading control. Bands were measured using densitometry and values first normalized to respective β-actin bands and then reported below each gel as relative to the vehicle-treated mice (longer exposure was used in the development of Du145 immunoblot). (B,C) mRNA were subjected to qRT-PCR for Notch pathway target genes (Hey1 and Hey2). β-actin was used as an internal control. (B: Du145, C: Du145R; One dot represents one mouse; * p<0.05,** p<0.01, NS=no significant difference).