Fluoview fv1000

Cells were grown on 24-well m-Slides (NEST Biotechnology Co. Ltd. Wuxi, China) and fixed with 4% paraformaldehyde for 30 min at 4°C, followed by treatment with 0.1% Triton for 10 min. The samples were blocked with PBST buffer (0.1% Tween-20) containing 10% goat serum at room temperature for 1 h and incubated with primary antibodies E-cadherin, N-cadherin (1 : 500, Epitomics, USA), and cytokeratin (1 : 200, Boster, China), overnight at 4°C. The cells then were washed in PBST and incubated with DyLight 488- and DyLight 594-conjugated secondary antibodies (ZS-Bio) at room temperature in the dark for 1 h. Nuclei were counterstained with DAPI for 5 min. After being washed three times, the cells were maintained with 50% glycerin in PBS and observed by laser confocal microscopy (Fluoview FV1000; Olympus, Tokyo, Japan). Photographs were taken with a digital camera (Olympus Fluoview FV1000) attached to a microscope. Ten images (approximately 30 cells per field) were acquired in each group, and the quantification of gray value was analyzed with Olympus Fluoview software FV10-ASW 1.7.

PMNs were seeded into 24-well glass-bottomed black plates at 4 × 10⁵ cells/well and then incubated at 37 °C with 5% CO₂. Each experiment included three groups: blank control group, experimental group, and negative control group. Previous work has shown endonuclease activity in T. spiralis ESP [21 (link)]. To eliminate DNase activity in the ESP, 25 μM nuclease inhibitor aurintricarboxylic acid (ATA) was added along with different concentrations of Ad3 ESP (2.5, 5, and 10 ng/μl) to the experimental group, and PMNs were pre-treated for 1 h. PMNs were then stimulated with 100 nM PMA for 3 h. After incubation, PMNs were stained with 5 µM Hoechst 33342 (Sigma, USA) and 5 µM SYTOX Green (Invitrogen, USA), and the samples were observed under a laser scanning confocal microscope (Olympus FluoView FV1000, Olympus America, Inc., USA). In addition, alterations in PMN morphology and nuclear dimensions were assessed by a laser scanning confocal microscope (Olympus FluoView FV1000, Olympus America, Inc., USA), and the nuclear expansion area for 500 cells in each sample well across the respective groups was quantified using ImageJ software (National Institutes of Health, USA).

The pCambia1300 vector was used to construct BiFC fusion vectors, which contained the N- or C-terminal of yellow fluorescence protein (YFP) fragments (YFPN and YFPC), respectively. The full-length CDSs of PperSLFLs without stop codon were cloned into pCambia1300-YFPN vectors, whereas the partial CDSs of PperS-RNase without stop codon and signal peptide were cloned into pCambia1300-YFPC. All the construct vectors were transformed into Agrobacterium tumefaciens GV3101 respectively and co-infiltrated into Nicotiana Benthamiana leaves. YFP fluorescence was observed in epidermal cell layers of N. Benthamiana leaves after infiltrated 5 days using Olympus BX61 fluorescent microscope (Olympus FluoView FV1000).
The box, box-V1, V1-V2, and HVa-HVb frames without the stop codon were cloned into the pCambia1300-YFPN vectors. The recombinant plasmids containing the box-YFPN, box-V1-YFPN, V1-V2-YFPN, or HVa-HVb-YFPN fusion gene and PperS1-RNase-YFPC, PperS2-RNase-YFPC, PperS2m-RNase-YFPC, or PperS4-RNase-YFPC without signal peptides fusion genes and the control plasmid with YFPN and YFPC were co-transformed into maize (Zea mays Linn.Sp.) protoplasts respectively according to Ren et al. (2011 (link)). YFP fluorescence was observed using Olympus BX61 fluorescent microscope (Olympus FluoView FV1000). The primers used were listed in Supplemental Table 2.