Fig 1.
Reovirus induces apoptosis in BrCa cell lines.
HTB133 and MCF7 cells were harvested at indicated time points following treatment with no virus (NV), dead virus (DV), or live virus (LV). Apoptosis was measured using flow cytometry via Annexin V binding, DNA fragmentation and Apo 2.7 expression. (N = 3, ± SD).
Fig 2.
Caspase 3/7 is activated during reovirus induced apoptosis of BrCa cells.
BrCa cells were infected with No virus (NV), LV and DV at a MOI of 40 PFU/cell, or untreated (NV). Cells were harvested at 72h and in situ active caspase 3/7 activity was measured using flow cytometry.
Table 1.
Transcriptional upregulation of apoptotic genes in HTB 133 and MCF breast cancer cells post reovirus infection*.
Fig 3.
Reovirus upregulates NF-kB activity in BrCa cells.
(A) NF-kB p65 mRNA expression is induced in reovirus infected BrCa cells. HTB133 and MCF7 cells were infected with 40 MOI of LV or DV for 12 and 24 hours. Total RNA extracted was assessed for NF-kB p65 and 18S gene expression by qPCR. Bars represent fold increase over untreated controls. (N = 2, ± SD). (B) Reovirus up regulates nuclear translocation of NF-kB in BrCa cells. BrCa cells were incubated with 40 MOI of reovirus for 12 hours and nuclear and cyto solic extracts were prepared. NV or LV treated proteins were subjected to SDS/PAGE and blotted with anti NF-kBp65, actin and histone antibody.
Fig 4.
Functional aptitude of reovirus activated NK-kB in BrCa.
(A) Reovirus induces NF-kB DNA binding in BrCa cells. BrCa cells were treated with either NV or LV for 12 hours or TNF (20 ng/ml) and nuclear extracts were prepared. 5 micro grams of nuclear proteins was incubated with an IR-dye labeled oligonucleotide consisting of the NF-kB consensus binding sequence and resolved by acrylamide gel electrophoresis and scanned in a LI-COR gel scanner. (B) NF-kB super shift complex is made up of p50 and p65. Nuclear proteins were incubated with 2 μl of super shift antibodies directed against NF-kB p50 or p65 before incubation with the NF-kB p65 probe. The mixtures were resolved in acrylamide gels and scanned as described under Fig 4A.
Fig 5.
Reovirus induces DNA binding of NF-kB in BrCa cells in a time dependent manner.
BrCa cells were treated with either NV or LV or TNF (20 ng/ml) and nuclear extracts were prepared at the times indicated. EMSAs were conducted as previously and scanned.
Fig 6.
Pharmacologic inhibition of NF-kB leads to suppression of reovirus induced cell death in BrCa cells.
MCF7 and HTB 133 cells were pre-incubated with either the NF-kB specific inhibitors CAPE (20 μM), or the proteasome inhibitor ALLN (10 μM), for 3 hours prior to reovirus infection. Cell death was assessed 24, and 48h via the WST assay (N = 5, ± SD). (*), significantly different.
Fig 7.
Reovirus infection of BrCa leads to upregulation of PUMA.
(A) PUMA mRNA expression is induced in reovirus infected BrCa cell lines. MCF7 and HTB 133 cells were infected with 40 MOI of reovirus for 12, 24 and 48 hours. PUMA gene expression was quantified by real-time PCR. Bars represent fold increase over untreated control cultures (N = 3, ± SD). (B) Reovirus treatment up regulates PUMA protein expression in BrCa cells. BrCa cell lines were incubated with 40 MOI of reovirus for 12 hours and cytosolic extracts were prepared. 50 μg of virus treated (LV) or untreated (NV) proteins were subjected to SDS/PAGE and blotted with anti PUMA antibody and HRP linked secondary antibody.
Fig 8.
Pharmacologic inhibition of NF-kB down regulates reovirus-induced PUMA gene expression in BrCa cells.
Real time PCR was performed on RNA isolated from MCF7, HTB133 and HTB30 cells incubated with either media alone, CAPE (20μM), ALLN (10μM) for 3 hours before incubation with 40 MOI reovirus for 12 hours. Bars represent fold increase over untreated control cultures (N = 3, ± SD).
Fig 9.
Molecular inhibition of PUMA leads to oncolytic protection.
(A) PUMA down regulation via siRNA leads to suppression of PUMA protein expression. HTB 133 cells were transfected with siRNA sequences against PUMA (Ambion) and total protein extracted was subjected to western analysis against PUMA and actin antibodies. (B) Oncolytic protection by PUMA down regulation. HTB 133 and MCF7 cells were transfected with siRNA sequences #2 against PUMA and infected with live or dead reovirus. Cells death was assayed via trypan blue (N = 3, ± SD).
Fig 10.
PUMA down regulation leads to modest apoptosis inhibition.
MCF7 and HTB 133 cells were transfected with on-target plus siRNA sequences against PUMA or control siRNA and infected with LV. Left panels -Total cellular proteins extracted were subjected to SDS PAGE and blotted against PUMA and actin antibodies. Right panels—HTB 133 and MCF7 cells were transfected with control or PUMA siRNA and infected with LV. Apoptosis was assayed via flow cytometry using annexin V/7AAD, (N = 3, ± SD).