Ed using a BioTek microplate reader (BioTek, Winooski, VT, USA). Pro-apoptotic effects were quantified by PI staining and FACS analysis of sub-G0/G1 DNA content as previously described.45 siRNA experiments. Caspase-4 and non-target SMARTpool siRNA were obtained from Dharmacon (Lafayette, CO, USA). Cells were transfected with 100 nM of each siRNA using Oligofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Transfected cells were incubated for 24 h and then treated with Reolysin, BZ, or the combination for 48 h. Efficiency of caspase-4 knockdown was measured at 48 h by immunoblotting. Quantitative real-time polymerase chain reaction. cDNA from Reolysin- or BZ-treated cells was used for relative quantification by RT-PCR analyses. cDNA synthesis was performed from 1 mg RNA in a 20-ml reaction mixture using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). CHOP (DDIT3), GADD34 (PPP1R15A), GRP78/BiP (HSPA5), calreticulin (CALR), PDI (P4HB), ERp57 (PDIA3), and GAPDH or b-actin transcripts were amplified using commercially available TaqMan Gene expression assays (Applied Biosystems). XBP-1s qRT-PCR was designed to span the 26-bp intron to anneal only the spliced mRNA. Primers and TaqMan probe sequences are as follows: forward primer: 50 -cctggttgctgaagaggag30 ; reverse primer: 50 -agtcaataccgccagaatcc-30 ; probe: 50 (Fam)-cctgcacctgctgcggactc-30 (Tamra). Relative gene expression was calculated with the 2 DDCt method using GAPDH as a housekeeping gene. Measurement of intracellular Ca2 levels. Panc-1 and CFPAC-1 cells were treated with Reolysin, BZ, or both for 16 h. Cells were collected, washed in PBS, and incubated with 1 mmol/l calcium green-1 (Invitrogen) for 30 min. Fluorescence was quantified using a FACSCanto II with CellQuest Pro Software (BD Biosciences, San Jose, CA, USA). Implantation of tumor cells and treatment schedule. Panc-1 pancreatic cancer cells were harvested from culture flasks and transferred to serum-free HBSS. Tumor cells (1 107 cells) were injected into the right flank of female nude mice and allowed to establish tumors. Following tumor formation, animals were pair matched by tumor size and placed into groups of eight mice. Animals were then treated by i.v. injection of 0.5 mg BZ per kg every 72 h, 5 108 TCID50 Reolysin once a week, or both agents for 5 weeks. Tumor volume and animal weight measurements were recorded twice weekly.Pemetrexed disodium Tumor tissue was collected for immunohistochemistry (IHC) and electron microscopy at the end of the study.BMS-986278 Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling assay.PMID:35567400 DNA fragmentation in tumor samples was analyzed using an FITC-labeled terminal deoxyribonucleotide transferase-mediated dUTP Cell Death and DiseaseReovirus induces ER stress JS Carew et alnick end labeling (TUNEL) assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PI was used to counterstain the nucleus. Images were captured with an Olympus fluorescent microscope (Olympus) with a DP71 camera and a 20 objective. Percentages of TUNEL-positive cells were determined by manual counting of five random fields per section. Immunohistochemistry. Paraffin-embedded tumor sections were deparaffinized in xylene and a graded series of alcohol and rehydrated in PBS. Heat-induced epitope retrieval on paraffin-embedded sections was performed by microwaving slides in a citrate buffer for 5 min. Endogenous peroxides were b.