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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.
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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.
NFB is retained in the cytoplasm in the presence of an
IBdominant 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
IBdominant 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 IBαa 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.
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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.