2. Materials and Methods
2.1 Ethics statement
In this study, animal experiments were not performed. All the samples were collected post-mortem from slaughterhouses, and therefore, no ethical approval was required.
2.2 Animal samples
Bladder mucosa samples from 15 cows clinically suffering from chronic enzootic hematuria were collected from public slaughterhouses after bladder neoplasms had been identified during mandatory post-mortem examination. Bladder mucosa samples from 10 healthy cows were also collected. Animals from both groups were 3-5 years old. All bladder samples were immediately subdivided and either fixed in 10% buffered formalin for microscopic investigation or frozen in liquid nitrogen and stored at – 80 °C for subsequent molecular biology analysis.
2.3. Antibodies
Rabbit antibodies against RIG-1, MDA5, IRF3, phospho-IRF3, TBK1, phospho-TBK1, and TRIM25, were obtained from Cell Signaling Technology (Leiden, Netherlands). Rabbit antibody anti-RNF135 (RIPLET) was purchased from Sigma-Aldrich (MO, USA). Mouse anti-MAVS, anti-Sec13, and β-actin antibodies were purchased from Santa Cruz Biotechnology (TX, USA). Rabbit polyclonal anti-E5 serum recognising the C-terminal 14 amino acids of the BPV E5 oncoprotein was kindly gifted provided by Prof. DiMaio (Yale University, New Haven USA).
2.4. RNA extraction and reverse transcription (RT)-PCR
Total RNA was extracted from bladder samples from 15 cows suffering from chronic enzootic hematuria and 10 healthy cows using the RNeasy Mini Kit (Qiagen, NW, DE), according to the manufacturer’s instructions. Genomic DNA was removed from the RNA preparations using RNase-free DNase Fermentas Life Sciences (Thermo Fisher Scientific, MA, USA). A total of 1 μg RNA was used to generate single-stranded cDNA, using the QuantiTect Reverse Transcription Kit (Qiagen NW, DE), according to the manufacturer’s instructions. PCR was performed with a specific primer set designed using Primer3, an online tool, for BPV-2 E5, BPV-13 E5, bovine RIG-I, MDA5, and TRIM25 genes. The following primers were used: BPV-2 E5ORF forward 5′-CACTGCCATTTGTTTTTTTC-3′, reverse 5′-GGAGCACTCAAAATGATCCC-3′; BPV-13 E5 ORF forward 5′-CACTGCCATTTGGTGTTCTT-3′, reverse 5′- AGCAGTCAAAATGATCCCAA-3′;RIG-I forward 5’- AGGAAAAGATTCGCCAGATACAGA-3’; reverse 5’-ATGGCATTCCTCCACCACTC-3’; MDA5 forward 5’-TGAAGCAGGGGTAAGAGAGC-3’; reverse 5’-TCAGACTCTGTACTGCCTTCAC-3’:TRIM25 forward 5’- CGGAGCTCCTGGAGTATGTG-3’; reverse 5’- TAGTTCAGGGATGCGTCAGC -3’. Conditions for PCR were as follows: 94 °C for 5 min, 40 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s.
2.5. Sequence analysis
PCR products, obtained by RT-PCR, were purified using the QIAquick PCR Purification Kit 131 (Qiagen NW, DE) and were subjected to bidirectional sequencing using the Big Dye-Terminator v1.1 Cycle 132 Sequencing Kit (Applied Biosystems, CA, USA), according to the manufacturer’s recommendations. Dye terminators from 133 sequences were removed using a DyeEx-2.0 Spin Kit (Qiagen), and sequences were run on a SeqStudio 134 Genetic Analyzer (Thermo Fischer Scientific, CA, USA). Electropherograms were analysed using Sequencing Analysis v5.2 and Sequence Scanner v1.0 softwares (Thermo Fischer Scientific, CA, USA). The sequences were analysed using the BLAST program.
2.6. Real-time reverse transcription PCR (RT-PCR).
To perform real-time RT-PCR analysis, total RNA and cDNA from diseased and healthy urinary bladder samples were generated, as described above. Real-time PCR was performed with a Bio-Rad CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), using iTAq Universal SYBR® Green Supermix (Bio-Rad). Each reaction was performed in triplicate, and the primers used for RIG-I, MDA5, and TRIM 25 were the same as those used for RT-PCR. The PCR thermal profile was as follows: 95 °C for 10 min, 40 cycles of 94 °C for 15 s, and 58 °C for 30 s, followed by a melting curve. Relative quantification (RQ) was calculated using the CFX Manager™ software, based on the equation RQ=2−ΔΔCq, where Cq is the quantification cycle to detect fluorescence. Cq data were normalised to the bovine β-actin gene (forward: 5′- TAGCACAGGCCTCTCGCCTTCGT-3′, reverse5′-147 GCACATGCCGGAGCCGTTGT-3′).
2.7.Western blot analysis
Healthy and diseased bovine urothelial samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 1% Triton X-100, 400 mM NaCl, 1 mM 151 ethylenediaminetetraacetic acid, 2 mM phenylmethylsulfonyl fluoride, 1.7 mg/mL aprotinin, 50 mM 152 NaF, and 1 mM sodium orthovanadate). Protein concentration was measured using the Bradford assay (Bio-Rad). For western blotting, 50 μg protein lysate was heated at 90 °C in 4X premixed Laemmli sample buffer (Bio-Rad), clarified by centrifugation, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (GE Healthcare, UK). Membranes were blocked with Tris-buffered saline and 0.1% Tween 20 (TBST)- containing 5% bovine serum albumin (BSA) for 1 h at room temperature. The membranes were subsequently incubated overnight at 4 °C with primary antibodies, washed three times with TBST, incubated for 1 h at room temperature with goat anti-rabbit or goat anti-mouse (Bio-Rad) HRP- conjugated secondary antibody, diluted at 1:5,000 in TBST containing 5% BSA, and washed three times with TBST. Immunoreactive bands were detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and ChemiDoc XRS Plus (Bio-Rad). Images were acquired using Image Lab Software version 2.0.1.
2.8. Immunoprecipitation
Total protein extracts from normal and pathological bladders, obtained as previously described, were immunoprecipitated. Protein samples (1mg) were incubated with anti-TRIM25 or anti-rabbit IgG (isotype), anti-Riplet or anti-rabbit IgG (isotype), and anti-MAVS or anti-mouse IgG antibodies (Bethyl Laboratories, Inc., TX, USA) for 1 h at 4 °C with gentle shaking. Thereafter, the samples were centrifuged at 1,000g for 5 min at 4 °C and incubated with 30 μL of Protein A/G-Plus Agarose (sc-184 2003) (Santa Cruz Biotechnology) overnight at 4 °C. The immunoprecipitates were washed four times in complete lysis buffer and separated on polyacrylamide gels. Subsequently, the proteins were transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature (25 °C) in TBST with 5% BSA, and then incubated with primary antibodies overnight at 4 °C. After three washes in TBST, the membranes were incubated with secondary antibodies for 1 h at room temperature. Chemiluminescent signals were developed using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and were detected using the ChemiDoc XRS gel documentation system (Bio-Rad).
2.9. Statistical analysis
Results are presented as the mean ± standard error (SE). Data were assessed by one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons of means using the GraphPad PRISM software version 8 (GraphPad Software, San Diego, CA, USA). A p-value ≤ 0.05 indicated statistical significance.