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Supplemental Information: Supplemental Figure 1. RB status does not impinge on the alterations of cell cycle after radiation. Actively growing LNCaP and LAPC4 cells were treated with radiation and cells were harvested and processed for further analysis. (A) Western blotting analysis of p21Cip1, p53 and a loading control laminB in hormone dependent RB proficient and RB deficient LNCaP cells after different time points post IR (10Gy). (B) Western blotting analysis of p21Cip1, p53 and a loading control laminB in hormone dependent RB proficient and RB deficient LAPC4 cells at indicated time points post IR (10Gy). (C) Graphic representation of BrdU incorporation in hormone dependent RB proficient and RB deficient LNCaP cells at different time points post IR (10Gy). (D) Graphic representation of BrdU incorporation in hormone dependent RB proficient and RB deficient LAPC4 cells at different time points post IR (10Gy). (E) Flow traces of BrdU incorporation in shCon LNCaP and shRB LNCaP cells at 6 and 24 hours following exposure to 0 or 10 Gy of ionizing radiation. (F) Flow traces of BrdU incorporation in shCon LNCaP and shRB LAPC4 cells at 6 and 24 hours following exposure to 0 or 10 Gy of ionizing radiation. (G) Western blot analysis of CDC25A and CDK2 in shCon and shRB LNCaP and LAPC4 cells exposed to 0 Gy or 10 Gy of ionizing radiation. For each data point is a mean ± SD from three or more independent experiments. ★★ p < 0.05 were considered as statistically significant. Supplemental Figure 2. RB deficiency promotes enhanced apoptotic pathway in response to ionizing radiation. (A) Actively growing LNCaP shCon and shRB cells were exposed to radiation therapy and processed for further analysis. Microarray analysis generated heat map of cell death and survival pathway genes from RB proficient and deficient LNCaP cells after 24 hours of post IR (10 Gy). ★★ p < 0.05 were considered as statistically significant. Supplemental Figure 3. NFB is retained in the cytoplasm in the presence of an IBdominant negative. (A) Graphic representation of NFκB p50 binding to consensus sequence by TF ELISA in RB deficient LNCaP cells with and without challenge by an IBdominant negative in the presence of wild-type oligonucleotides and mutant oligonucleotides 24 hours post radiation therapy (10 Gy). (B) Graphic representation of NFκB p50 binding to consensus sequence by TF ELISA in RB deficient LAPC4 cells with and without challenge by an IBαa dominant negative in the presence of wild-type oligonucleotides and mutant oligonucleotides 24 hours post radiation therapy (10 Gy). immunoblot analysis of PLK3, cleaved caspase3 and laminB in PLK3 deficient LNCaP shRB cells. Each data point is a mean ± SD from three or more independent experiments. ★★ p < 0.05 were considered as statistically significant over Control. Supplemental Figure 4. PLK3 expression promotes apoptotic cell death in the setting of RB loss. (A) Cell growth and immunoblot analysis of PLK3, cleaved caspase 3 and laminB in LAPC4 shCon or shRB cells expressing PLK3 cDNA. (B) Cell growth and immunoblot analysis of PLK3, cleaved caspase 3 and lamin B in PLK3 deficient LAPC4 shRB cells in response to radiation (10Gy immunoblot analysis of PLK3, cleaved caspase3 and laminB in PLK3 deficient LNCaP shRB cells. Each data point is a mean ± SD from three or more independent experiments. statistically significant over control. ★★ p < 0.05 were considered as Supplemental Figure 5. RB status is retained in biopsy proven local recurrence. Immunohistochemical analysis of pRB in recurrent human prostate tumors treated with prior definitive radiation therapy.