Newcastle disease virus-induced caspase-independent apoptosis pathway in BHK-21 cells

Chen-Wei Wang Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology Chia-Ying Lin Da Dian Biotechnology Company Limited Sheng-Chang Chung Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology Wen-Ling Shih Department of Biological Science and Technology, National Pingtung University of Science and Technology Tzu-Chieh Lin International Degree Program in Animal Vaccine Technology, National Pingtung University of Science and Technology Duangsuda Thongchan Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin campus hung-yi wu (  wuhy@g4e.npust.edu.tw ) National Pingtung University of Science and Technology Department of Veterinary Medicine Department of Veterinary Medicine https://orcid.org/0000-0001-5748-8105

added, and the sample was stored at −80°C overnight. The sample pellet 143 was collected and washed with cold 80% ethanol. The samples were dried 144 at room temperature for 10 min, dissolved in deionization and distilled 145 water, and diluted to a concentration of 5 μg per well. Finally, 146 electrophoresis was performed using 2% agarose gel. 147 148 TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end 149 labeling assay) 150 The BHK-21 cells were diluted at a concentration of 3 × 10 5 and infected 151 with NDV MOI of 1. The sample was separated into the caspase and 152 caspase inhibitor groups. After the 0, 12, and 24 h of incubation at 37°C 153 in an atmosphere of 5% CO 2 , the TUNEL assay was carried out using a 154 FragELTM DNAFragmentation Detection Kit (CALBIOCHEM,155 QIA33-1EA). The cover slip was sealed with 40% glycerol, and 156 observation and counting were done under a microscope. 157 158

caspase-independent pathway in BHK-21 cells 214
The NDV-treated BHK-21 cells had been divided into two groups, one 215 group was directly challenge by NDV, and the other group was treated 216 with pan-caspase inhibiter z-VAD-fmk (final concentration 100 μM) to 217 inhibit caspase-dependent pathway apoptosis before challenged with 218 NDV. Cell apoptosis was induced by a caspase-dependent pathway with 219 activation of caspase-3 by entering the nuclei; the caspase protein will cut 220 the DNA according to nucleosome nucleotides length, causing DNA 221 fragmentation. In Figure 1a, the caspase group indicated that the caspase 222 family protein cut the DNA according to nucleosome nucleotides length 223 that became the DNA markers in the 2% agarose gel. The caspase 224 inhibition group did not take the shape of DNA ladders after NDV 225 infection, as the caspase group did (Figure 1b). The rate of cell apoptosis 226 and cells fusion increased as time passed. The ratios of apoptosis of the 227 caspase group from 0, 12, and 24 h were 6.50 ± 1.69%, 29.59 ± 3.52%, 228 and 62.48 ± 3.69%, respectively. On the other hand, for the caspase 229 inhibitor group, the apoptosis ratios from 0, 12, and 24 h were 5.94 ± 230 0.75%, 22.92 ± 1.66%, 32.57 ± 1.31%, respectively. The data are shown 231 in Figure 3a-g. In addition, we observed that NDV infection increased 232 cell fusion depending on the infection time. The ratios of cell fusion of 233 caspase group at 0, 12, and 24 h were 0 ± 0%, 7.15 ± 1.32%, and 35.81 ± 234 3.53%, respectively, and in caspase inhibitor group, the ratios of cell 235 fusion at 0, 12, and 24 h were 0 ± 0%, 4.85 ± 2.77%, and 17.55 ± 1.84%, 236 respectively (Fig 2h). 237

NDV mediates BHK-21 cell apoptosis caspase-dependent and 239
-independent pathway. 240 NDV induced apoptosis via caspase-dependent and caspase-independent 241 pathways in BHK-21 cells. In the two groups, we used 0 h protein as a 242 starting point to confirm protein expression at 12 and 24 h. The results 243 reveal that the proapoptotic proteins Bax, cytochrome C, Mst3, and AIF 244 were increased over time and the antiapoptotic protein Bcl-2 decreased 245 over time, regardless of the caspase-dependent or -independent pathway. 246 Among them, Endo G was special; it was increased at 24 h in the 247 caspase-dependent pathway and increased over time in the 248 caspase-independent pathway; however, only the cleaved caspase-3 was 249 different. It was inhibited by z-VAD-fmk in the caspase-independent 250 pathway. In addition to apoptosis, we found that NDV-treated BHK-21 251 cells had less actin, regardless of having caspase or not. The actin always 252 decreased in the NDV-treated BHK-21 cells at 12 and 24 h. This was 253 because we used GAPDH to do protein internal control, not used actin. 254 The western blot data were shown in Figure. 3. 255

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In apoptosis caspase-independent pathway, Mst3, AIF, and Endo G are 257 transferred from the mitochondria to the nucleus 258 In Figure 4a and 4b, the sample was separated it two groups; one was 259 caspase apoptosis group that had 0 h total protein (0 h T), 0 h cytoplasmic 260 protein (0 h C), 0 h nucleus protein (0 h N), 24 h total protein (24 h T), 24 261 h cytoplasmic protein (24 h C), and 24 h nucleus protein (24 h N). The 262 other group were caspase inhibited group that had same protein samples. 263 The total protein internal control was actin, the cytoplasmic protein 264 sample was α-tubulin, and the nucleus protein sample was lamin B. In 265 Figure 4a, we confirm the transfer of Mst3, AIF, and Endo G from 266 cytoplasm to the nucleus; Figure 4b shows that the caspase inhibition 267 group was similar to the caspase group in terms of current protein 268 expression. After that, we showed the image data in immunofluorescent 269 staining in Figure 5a and 5b. In Figure 5a and 5b, the green color is target 270 protein (FITC), red color is mitochondria (Mitoracker), and blue marks 271 the nucleus (DAPI). The FITC three different target proteins were AIF, 272 Endo G, and NDV. In Figure 5a  NDV apoptosis papers showed that both the extrinsic and intrinsic 295 pathways are activated in NDV-induced apoptosis. In 2011 the paper 296 confirmed that PC12 rat pheochromocytoma cell mitogen-activated 297 protein kinase pathway was not involved in NDV-induced apoptosis, but 298 in A549 human lung cancer, can induce apoptosis using the p38 and 299 MAPK pathway, in addition, the endoplasmic reticulum apoptosis 300 pathway no effect [17,20]. When NDV induces cell apoptosis, Bax 301 transfers from cytoplasm to mitochondria and cytochrome C transfers 302 from mitochondria to cytoplasm. But Bcl-2 of protein expression is not 303 any increase and decrease in NDV-induced apoptosis. If the caspase 304 would be suppressed, the cell would be induced caspase-independent 305 apoptosis pathway [16,18,21]. In summary, NDV-induced apoptosis has 306 two mechanisms; one is NDV has different apoptosis pathways induced 307 by different cells, and there is no absolute path followed. The other is 308 NDV-induced apoptosis is a complex multiple pathway response. Our 309 study showed that cytochrome C, AIF, Mst3, and Endo G were important 310 because their proteins express increasingly form 0 to 24 h. Bax in 311 caspase-dependent or independent pathway is increase protein expression, 312 but the Bcl-2 is decrease. 313

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The Bax protein configuration changes and aggregation can promote 315 apoptosis; Bcl-2 can prevent that when the Bcl-2 protein expressed more 316 than Bax and there is no apoptosis [22][23][24][25]. His study of NDV-induced 317 HeLa cell apoptosis showed that Bax configuration changed before NDV 318 infected the cell and that the Bax protein changed location and caused 319 cytochrome C from the mitochondria to be released into the cytoplasm; In this study, we show the NDV-mediated BHK-21 cell apoptosis 366 mechanisms involve complex pathway; when in the normal state, the 367 caspase-dependent pathway is main apoptosis pathway; when the caspase 368 is suppressed, the BHK-21 can switch on the caspase-independent 369 pathway by AIF, Endo G, or Mst3 to allow apoptosis to continue. And 370 actin will also be affected by caspase, which may explain the lower actin 371 content over time. Endo G, and cleaved caspase-3, the antiapoptotic protein Bcl-2, internal 582 control GADPH, and actin. 583 (3a) After BHK-21 treatment with NDV for 0, 12, and 24 h, the 584 caspase-dependent apoptosis-associated proteins were expression. 585 (3b) After BHK-21 treatment with NDV for 0, 12, and 24 h, the 586 caspase-independent apoptosis-associated proteins were expression. 587 (3c) The graph represents protein expression in the caspase-dependent 588 group. The x-axis represents apoptosis-associated proteins and the y-axis 589 represents the relative fold expression, challenge time as 0, 12, and 24 h. 590 (3d) The graph represents protein expression in the caspase inhibition 591 group. The x-axis represents apoptosis-associated proteins and the y-axis 592 represents the relative fold expression, challenge time as 0, 12, and 24 h. The 2% gel electrophoresis of genomic DNA from BHK-21 cell challenged with NDV. M is the 100 bp marker. Lanes 1, 2, and 3 represent the extraction time of DNA at 0, 12, and 24 h after NDV challenge. Figure 1a shows the caspase group and 1b shows the caspase inhibition group. . 2g and 2h. The trend of apoptosis rate in two groups of BHK-21 cells under different time periods of NDV challenge, the increasing tendency of apoptosis and cell fusion with or without addition of caspase inhibitor z-VAD-fmk is con rmed by TUNEL assay. Figure 2g x-axis represents time after NDV challenge while y-axis is percentage of cells undergoing apoptosis. Results are shown in the form of mean value ± SD for the three experiments. Figure 2h is the x-axis representing time after NDV challenge while y-axis stands for percentage of cell undergoing fusion. The results are shown in the form of mean value ± SD for the three experiments.  The 2% gel electrophoresis of genomic DNA from BHK-21 cell challenged with NDV. M is the 100 bp marker. Lanes 1, 2, and 3 represent the extraction time of DNA at 0, 12, and 24 h after NDV challenge. Figure 1a shows the caspase group and 1b shows the caspase inhibition group. . 2g and 2h. The trend of apoptosis rate in two groups of BHK-21 cells under different time periods of NDV challenge, the increasing tendency of apoptosis and cell fusion with or without addition of caspase inhibitor z-VAD-fmk is con rmed by TUNEL assay. Figure 2g x-axis represents time after NDV challenge while y-axis is percentage of cells undergoing apoptosis. Results are shown in the form of mean value ± SD for the three experiments. Figure 2h is the x-axis representing time after NDV challenge while y-axis stands for percentage of cell undergoing fusion. The results are shown in the form of mean value ± SD for the three experiments. Figure 3