Rnaase

Cells were collected and washed using PBS three times after compound or DMSO treatment on different days according to the requirements. For cell cycle detection, cells were fixed by 70% EtOH overnight at 4 ^oC. PI solution (200 μL) containing RNAase (Thermo) was added, the mixture was incubated for 15 min, then samples were filtered (40–50 μm nylon net) before detection. FITC Annexin V-PI Apoptosis Detection Kit I (BD Biosciences, 556547) was used for apoptosis detection. The procedures were as follows: 50 μL binding buffer, 2.5 μL PI, and 2.5 μL Annexin V were added to resuspended samples for 15 min. Then 150 μL binding buffer were added before centrifugation. Finally, the cells were resuspended in 200 μL binding buffer for FCM analysis. For CD11b (BD Biosciences, 555388), CD14 (BD Biosciences, 555397), CD41b (BD Biosciences, 555469), and CD61 (BD Biosciences, 555754) detection, antibodies were added to cells prior to incubation for 30 min, then they were washed by PBS three times before FCM detection. A mitochondrial membrane potential detection kit (BD Biosciences, USA) was selected for mitochondrial membrane potential detection and used according to the manufacturer’s instructions. FCM assays were conducted on FCM (ACEABIO | NovoCyte or BD FACSCanto, USA). Repeat three times for each sample.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, trypsin, and crystal violet (CV) were purchased from Sigma-Aldrich (St Louis, MO, USA). Dimethylsulphoxide (DMSO), ethanol, and acetic acid were purchased from Merck (Darmstadt, Germany). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). DAPI, PI, and RNAase were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
P. ecklonii Benth air-dried and powdered plant material was given by the Faculty of Pharmacy of the University of Lisbon and it was collected from seeds provided by the herbarium of the National Botanical Garden of Kirstenbosch, South Africa. Voucher specimens (S/No. LISC) have been deposited in the herbarium of the Tropical Research Institute in Lisbon [11 (link)]. Acetone and other organic solvents were from analytic grade and provided by (VWR international S.A.S., Briare, France); Silica for the isolation was obtained from Merck (grade 60, 230–400 mesh, Merck KGaA, Darmstadt, Germany).

Total DNA from N. benthamiana was isolated using CTAB reagent and treated with RNAase (Thermo scientific, USA). DNA was purified by Phenol: Chloroform: Isoamyl (PCI) extraction. For southern blotting, ten micrograms of total plant DNA was loaded on to 0.8% 1xTAE agarose gel and transferred to nitrocellulose membrane⁶³ . For detection of viral DNA, coat protein gene was used as probe and labeled with DIG-High Prime DNA labeling kit (Roche diagnostics, Germany). Membrane was hybridized with probe and detection was performed by CDP-star (Roche diagnostics, Germany). Protein extraction was done by pulverizing the leaves in liquid nitrogen and homogenized to fine powder. To this, 200 µl protein extraction solution (40 mM tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH-7.4, 300 mM sodium chloride (NaCl), 5 mM magnesium chloride (MgCl₂), 2 mM ethylenediaminetetraacetic acid (EDTA), 4 mM dithiothreitol (DTT), 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5% Glycerol and protease inhibitor cocktail) was added, samples were boiled for 5 minutes and subsequently loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed protein samples were transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA) and detection was done by AV2 antiserum.